Chemotherapeutic Drug-Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA.

PubMed

Lin, Kevin N; Grandhi, Taraka Sai Pavan; Goklany, Sheba; Rege, Kaushal

2018-04-10

Plasmid DNA (pDNA) is an attractive therapeutic biomolecule in several diseases including cancer, AIDS, cystic fibrosis, Parkinson's disease, and Alzheimer's disease. Increasing demand for plasmid DNA as a therapeutic biomolecule for transgene expression or vaccine applications necessitate novel approaches to bioprocessing. The synthesis, characterization and evaluation of aminoglycoside-derived hydrogel microbeads (Amikabeads) for pDNA binding is described previously. Here, the generation and evaluation of novel chemotherapeutic drug-conjugated microbeads for application in pDNA binding and recovery is described. Chemotherapeutic drug-conjugated Amikabeads demonstrate higher binding of methylated pDNA compared to unmethylated pDNA in presence of high salt concentrations. Desorption of plasmids from drug-conjugated microbeads is facilitated by the use of organic modifiers. The observed differences in binding methylated versus unmethylated DNA can make drug-conjugated microbeads useful in diagnostic as well as therapeutic applications. These results demonstrate that anti-cancer drugs represent a diverse set of ligands that may be exploited for molecular engineering of novel DNA binding materials for applications in delivery, diagnostics, and biomanufacturing. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA sequence-selective C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates show anti-tubercular-specific activities.

PubMed

Brucoli, Federico; Guzman, Juan D; Basher, Mohammad A; Evangelopoulos, Dimitrios; McMahon, Eleanor; Munshi, Tulika; McHugh, Timothy D; Fox, Keith R; Bhakta, Sanjib

2016-12-01

New chemotherapeutic agents with novel mechanisms of action are in urgent need to combat the tuberculosis pandemic. A library of 12 C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-heterocyclic polyamide conjugates (1-12) was evaluated for anti-tubercular activity and DNA sequence selectivity. The PBD conjugates were screened against slow-growing Mycobacterium bovis Bacillus Calmette-Guérin and M. tuberculosis H 37 Rv, and fast-growing Escherichia coli, Pseudomonas putida and Rhodococcus sp. RHA1 bacteria. DNase I footprinting and DNA thermal denaturation experiments were used to determine the molecules' DNA recognition properties. The PBD conjugates were highly selective for the mycobacterial strains and exhibited significant growth inhibitory activity against the pathogenic M. tuberculosis H 37 Rv, with compound 4 showing MIC values (MIC=0.08 mg l -1 ) similar to those of rifampin and isoniazid. DNase I footprinting results showed that the PBD conjugates with three heterocyclic moieties had enhanced sequence selectivity and produced larger footprints, with distinct cleavage patterns compared with the two-heterocyclic chain PBD conjugates. DNA melting experiments indicated a covalent binding of the PBD conjugates to two AT-rich DNA-duplexes containing either a central GGATCC or GTATAC sequence, and showed that the polyamide chains affect the interactions of the molecules with DNA. The PBD-C8 conjugates tested in this study have a remarkable anti-mycobacterial activity and can be further developed as DNA-targeted anti-tubercular drugs.

Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery.

PubMed

Bahadoran, Azadeh; Moeini, Hassan; Bejo, Mohd Hair; Hussein, Mohd Zobir; Omar, Abdul Rahman

In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-).  In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine.  The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Effects of Different Buffers on the Construction of Aptamer Sensors

NASA Astrophysics Data System (ADS)

Yu, Quan; Dai, Zhao; Wu, Wenjing; Zhu, Haijia; Ji, Luyu

2017-12-01

In this paper, the effect of different buffers, PBS and TBE, on the construction of an aptamer sensor (apt sensor) for ATP was investigated. The apt sensor was based on fluorescence energy resonance transfer (FRET), when the energy donor was 5'-carboxyfluorescein (5'-FAM) and the energy receptor was Au nanoparticles (AuNPs), respectively. Both the donor and acceptor were conjugated with complementary and single stranded DNA (ssDNA). The fluorescent changes of the sensors were measured to investigate the influence of different buffers during the whole preparation and detection process. The results indicated that when the AuNPs and ssDNA (Au-DNA1) were conjugated in PBS buffer, the corresponding apt sensors would obtain a better detection ability of ATP than in TBE buffer.

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

DOE PAGES

Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian; ...

2017-07-24

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

Analytical Devices Based on Direct Synthesis of DNA on Paper.

PubMed

Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

2016-01-05

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

PubMed Central

Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2016-01-01

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLaughlin, K. J.; Nash, R. P.; Redinbo, M. R.

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. Inmore » this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.« less

DNA-programmable multiplexing for scalable, renewable redox protein bio-nanoelectronics.

PubMed

Withey, Gary D; Kim, Jin Ho; Xu, Jimmy

2008-11-01

A universal, site-addressable DNA linking strategy is deployed for the programmable assembly of multifunctional, long-lasting redox protein nanoelectronic devices. This addressable linker, the first incorporated into a redox enzyme-nanoelectronic system, promotes versatility and renewability by allowing the reconfiguration and replacement of enzymes at will. The linker is transferable to all redox proteins due to the simple conjugation chemistry involved. The efficacy of this linking strategy is assessed using two model enzymes, glucose oxidase (GOx) and alcohol dehydrogenase (ADH), self-assembled onto separate nanoelectrode regions comprised of a highly ordered carbon nanotube (CNT) array. The sequence-specificity of DNA hybridization provides the means of encoding spatial address to the self-assembling process that conjugates enzymes tagged with single-stranded DNA (ssDNA) to the tips of designated CNTs functionalized with the complementary strands. In this study, we demonstrate the feasibility of multiplexed, scalable, reconfigurable and renewable transduction of redox protein signals by virtue of DNA addressing.

A septal chromosome segregator protein evolved into a conjugative DNA-translocator protein

PubMed Central

Sepulveda, Edgardo; Vogelmann, Jutta

2011-01-01

Streptomycetes, Gram-positive soil bacteria well known for the production of antibiotics feature a unique conjugative DNA transfer system. In contrast to classical conjugation which is characterized by the secretion of a pilot protein covalently linked to a single-stranded DNA molecule, in Streptomyces a double-stranded DNA molecule is translocated during conjugative transfer. This transfer involves a single plasmid encoded protein, TraB. A detailed biochemical and biophysical characterization of TraB, revealed a close relationship to FtsK, mediating chromosome segregation during bacterial cell division. TraB translocates plasmid DNA by recognizing 8-bp direct repeats located in a specific plasmid region clt. Similar sequences accidentally also occur on chromosomes and have been shown to be bound by TraB. We suggest that TraB mobilizes chromosomal genes by the interaction with these chromosomal clt-like sequences not relying on the integration of the conjugative plasmid into the chromosome. PMID:22479692

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

PubMed

Paul, Atanu; Wang, Bin

2017-05-18

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

PubMed Central

Kim, Taejin; Chitteni-Pattu, Sindhu; Cox, Benjamin L.; Wood, Elizabeth A.; Sandler, Steven J.; Cox, Michael M.

2015-01-01

The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism. PMID:26047498

Unique helicase determinants in the essential conjugative TraI factor from Salmonella enterica serovar Typhimurium plasmid pCU1.

PubMed

McLaughlin, Krystle J; Nash, Rebekah P; Redinbo, Mathew R

2014-09-01

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Gel Electrophoresis of Gold-DNA Nano-Conjugates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pellegrino, T.; Sperling, R.A.; Alivisatos, A.P.

2006-01-10

Single stranded DNA of different lengths and different amounts was attached to colloidal phosphine stabilized Au nanoparticles. The resulting conjugates were investigated in detail by a gel electrophoresis study based on 1200 gels. We demonstrate how these experiments help to understand the binding of DNA to Au particles. In particular we compare specific attachment of DNA via gold-thiol bonds with nonspecific adsorption of DNA. The maximum number of DNA molecules that can be bound per particle was determined. We also compare several methods to used gel electrophoresis for investigating the effective diameter of DNA-Au conjugates, such as using a calibrationmore » curve of particles with known diameters and Ferguson plots.« less

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

PubMed

Mutti, Elena; Hunger, Miriam; Fedosov, Sergey; Nexo, Ebba; Kräutler, Bernhard

2017-11-16

The synthesis and structural characterization of Co-(dN) 25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN) 39 -Cbl, which are organometallic DNA-B 12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN) 25 -Cbl, or radiolabeled vitamin B 12 ( 57 Co-CNCbl) and Co-(dN) 25 -Cbl or Co-(dN) 39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN) 18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functionalisation of Detonation Nanodiamond for Monodispersed, Soluble DNA-Nanodiamond Conjugates Using Mixed Silane Bead-Assisted Sonication Disintegration

DOE PAGES

Edgington, Robert; Spillane, Katelyn M.; Papageorgiou, George; ...

2018-01-15

Here, nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functionalmore » silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.« less

Functionalisation of Detonation Nanodiamond for Monodispersed, Soluble DNA-Nanodiamond Conjugates Using Mixed Silane Bead-Assisted Sonication Disintegration.

PubMed

Edgington, Robert; Spillane, Katelyn M; Papageorgiou, George; Wray, William; Ishiwata, Hitoshi; Labarca, Mariana; Leal-Ortiz, Sergio; Reid, Gordon; Webb, Martin; Foord, John; Melosh, Nicholas; Schaefer, Andreas T

2018-01-15

Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.

Functionalisation of Detonation Nanodiamond for Monodispersed, Soluble DNA-Nanodiamond Conjugates Using Mixed Silane Bead-Assisted Sonication Disintegration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edgington, Robert; Spillane, Katelyn M.; Papageorgiou, George

Here, nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functionalmore » silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.« less

The role of polymerase III in conjugation between E. coli K12 donor and recipient strains carrying dnaE ts mutation.

PubMed

Blinkowa, A

1976-01-01

The possible role of DNA polimerase III in conjugation was studied in a series of mutants temperature-sensitive for DNA polymerase III synthesis. The temperature-sensitive DNA mutation called dnaE 486 (ts) prohibits vegetative DNA replication at 41-45 degrees. Transfer of episome and chromosome from temperature-sensitive donor, carrying dnaE mutation to wild-type recipient strains, revertants and dnaE recipients was investigated. In the first two cases the number of Lac+ sexductants being even slightly higher at 43 degrees. Conjugational synthesis accompanying transfer involving the combination of dnaE (ts) thymine dependent and thymine independent donor and recipient strains measured by incorporation of 14C thymine was observed at the restrictive temperature. In the case of conjugation with temperaturesensitive recipient strains a drop of Lac+ sexductants and Pro+ recombinants may be as a result of disturbances in the synthesis of complementary strand in recipient, known to be dependent on pol III. However, the episome investigated by centrifugation in neutral CsC1 gradient after its transfer to the recipient with faulty polymerase III was double stranded (replicated) at the restrictive temperature.

DNA-cell conjugates

DOEpatents

Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

2016-05-03

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

DNA-cell conjugates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

The role of interindividual variation in human carcinogenesis.

PubMed

Lai, C; Shields, P G

1999-02-01

The process of chemical carcinogenesis is a complex multistage process initiated by DNA damage in growth control genes. Carcinogens enter the body from a variety of sources, but most require metabolic activation before they can damage DNA. There are multiple protective processes that include detoxification and conjugation, DNA repair and programmed cell death. Most of these functions exhibit wide interindividual variation in the population and thus are thought to affect cancer risk. The role of gene-environment interactions is being explored, and current data indicate that genetic susceptibilities can modify carcinogen exposures from the diet and tobacco smoking, although much more data exist for the latter. This review addresses the relationships of human carcinogenesis to these interindividual differences of phase I, phase II and DNA repair enzymes.

Treatment with Cefotaxime Affects Expression of Conjugation Associated Proteins and Conjugation Transfer Frequency of an IncI1 Plasmid in Escherichia coli

PubMed Central

Møller, Thea S. B.; Liu, Gang; Boysen, Anders; Thomsen, Line E.; Lüthje, Freja L.; Mortensen, Sisse; Møller-Jensen, Jakob; Olsen, John E.

2017-01-01

Horizontal gene transfer (HGT) is the major mechanism responsible for spread of antibiotic resistance. Antibiotic treatment has been suggested to promote HGT, either by directly affecting the conjugation process itself or by selecting for conjugations subsequent to DNA transfer. However, recent research suggests that the effect of antibiotic treatment on plasmid conjugation frequencies, and hence the spread of resistance plasmids, may have been overestimated. We addressed the question by quantifying transfer proteins and conjugation frequencies of a blaCTX−M−1 encoding IncI1 resistance plasmid in Escherichia coli MG1655 in the presence and absence of therapeutically relevant concentrations of cefotaxime (CTX). Analysis of the proteome by iTRAQ labeling and liquid chromatography tandem mass spectrometry revealed that Tra proteins were significantly up-regulated in the presence of CTX. The up-regulation of the transfer machinery was confirmed at the transcriptional level for five selected genes. The CTX treatment did not cause induction of the SOS-response as revealed by absence of significantly regulated SOS associated proteins in the proteome and no significant up-regulation of recA and sfiA genes. The frequency of plasmid conjugation, measured in an antibiotic free environment, increased significantly when the donor was pre-grown in broth containing CTX compared to growth without this drug, regardless of whether blaCTX-M-1 was located on the plasmid or in trans on the chromosome. The results shows that antibiotic treatment can affect expression of a plasmid conjugation machinery and subsequent DNA transfer. PMID:29238335

Robust and specific ratiometric biosensing using a copper-free clicked quantum dot-DNA aptamer sensor

NASA Astrophysics Data System (ADS)

Zhang, Haiyan; Feng, Guoqiang; Guo, Yuan; Zhou, Dejian

2013-10-01

We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free ``click chemistry'' (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free ``click chemistry'' (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate. Electronic supplementary information (ESI) available: Details on the synthesis, purification and characterisation of the DHLA-PEG600-N3, cyclooctyne-DNA, and QD-TBA20 conjugates as well as all supporting figures and tables. See DOI: 10.1039/c3nr02897f

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

PubMed Central

Kumar, S; Reed, M W; Gamper, H B; Gorn, V V; Lukhtanov, E A; Foti, M; West, J; Meyer, R B; Schweitzer, B I

1998-01-01

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions. PMID:9443977

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

PubMed

Kumar, S; Reed, M W; Gamper, H B; Gorn, V V; Lukhtanov, E A; Foti, M; West, J; Meyer, R B; Schweitzer, B I

1998-02-01

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

PubMed

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

PubMed

Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

2016-08-16

Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

PubMed Central

Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

2012-01-01

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

Multiple pathways of plasmid DNA transfer in Helicobacter pylori.

PubMed

Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

2012-01-01

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.

Controlled assembly of artificial protein-protein complexes via DNA duplex formation.

PubMed

Płoskoń, Eliza; Wagner, Sara C; Ellington, Andrew D; Jewett, Michael C; O'Reilly, Rachel; Booth, Paula J

2015-03-18

DNA-protein conjugates have found a wide range of applications. This study demonstrates the formation of defined, non-native protein-protein complexes via the site specific labeling of two proteins of interest with complementary strands of single-stranded DNA in vitro. This study demonstrates that the affinity of two DNA-protein conjugates for one another may be tuned by the use of variable lengths of DNA allowing reversible control of complex formation.

DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

PubMed Central

Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

2002-01-01

The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, “real-time” DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated. PMID:12167673

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation.

PubMed

Miguel-Arribas, Andrés; Hao, Jian-An; Luque-Ortega, Juan R; Ramachandran, Gayetri; Val-Calvo, Jorge; Gago-Córdoba, César; González-Álvarez, Daniel; Abia, David; Alfonso, Carlos; Wu, Ling J; Meijer, Wilfried J J

2017-01-01

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer ( oriT ), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis . We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1 LS20 and aux2 LS20 , and which we show are essential for conjugation. Both Aux1 LS20 and Aux2 LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1 LS20 and Aux2 LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriT LS20 , although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1 LS20 and/or Aux2 LS20 are located upstream of almost 400 relaxase genes of the Rel LS20 family (MOB L ) of relaxases. Thus, Aux1 LS20 and Aux2 LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Oxazine dye-conjugated dna oligonucleotides: Förster resonance energy transfer in view of molecular dye-DNA interactions.

PubMed

Kupstat, Annette; Ritschel, Thomas; Kumke, Michael U

2011-12-21

In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive Förster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large Förster distance of ~7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.

The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine.

PubMed

Zhou, Xianfeng; Zhang, Xizhen; Yu, Xianghui; Zha, Xiao; Fu, Qiuan; Liu, Bin; Wang, Xueyun; Chen, Yan; Chen, Yue; Shan, Yaming; Jin, Yinghua; Wu, Yongge; Liu, Junqiu; Kong, Wei; Shen, Jiacong

2008-01-01

Nonviral gene delivery systems based on conventional high molecular weight chitosans are efficient as DNA vaccine delivery system, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow onset of action. Furthermore, Chitosan oligomers shorter than 14 monomers units were recently found to form only weak complexes with DNA, resulting in physically unstable polyplexes in vitro and in vivo. Here, low molecular weight chitosans with an average molecular mass of 6kDa (Chito6) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting Chito6-GNPs conjugates as vectors for the delivery of plasmid DNA has been investigated in vitro and in vivo. After delivery by intramuscular immunization in BALB/c mice, the Chito6-GNPs conjugates induced an enhanced serum antibody response 10 times more potent than naked DNA vaccine. Additionally, in contrast to naked DNA, the Chito6-GNPs conjugates induced potent cytotoxic T lymphocyte responses at a low dose.

DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

NASA Astrophysics Data System (ADS)

Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

2016-02-01

Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

NASA Astrophysics Data System (ADS)

Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

2016-05-01

Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

Supramolecular 1-D polymerization of DNA origami through a dynamic process at the 2-dimensionally confined air-water interface.

PubMed

Yonamine, Yusuke; Cervantes-Salguero, Keitel; Minami, Kosuke; Kawamata, Ibuki; Nakanishi, Waka; Hill, Jonathan P; Murata, Satoshi; Ariga, Katsuhiko

2016-05-14

In this study, a Langmuir-Blodgett (LB) system has been utilized for the regulation of polymerization of a DNA origami structure at the air-water interface as a two-dimensionally confined medium, which enables dynamic condensation of DNA origami units through variation of the film area at the macroscopic level (ca. 10-100 cm(2)). DNA origami sheets were conjugated with a cationic lipid (dioctadecyldimethylammonium bromide, 2C18N(+)) by electrostatic interaction and the corresponding LB-film was prepared. By applying dynamic pressure variation through compression-expansion processes, the lipid-modified DNA origami sheets underwent anisotropic polymerization forming a one-dimensionally assembled belt-shaped structure of a high aspect ratio although the thickness of the polymerized DNA origami was maintained at the unimolecular level. This approach opens up a new field of mechanical induction of the self-assembly of DNA origami structures.

DNA-nanoparticle assemblies go organic: macroscopic polymeric materials with nanosized features.

PubMed

Mentovich, Elad D; Livanov, Konstantin; Prusty, Deepak K; Sowwan, Mukules; Richter, Shachar

2012-05-30

One of the goals in the field of structural DNA nanotechnology is the use of DNA to build up 2- and 3-D nanostructures. The research in this field is motivated by the remarkable structural features of DNA as well as by its unique and reversible recognition properties. Nucleic acids can be used alone as the skeleton of a broad range of periodic nanopatterns and nanoobjects and in addition, DNA can serve as a linker or template to form DNA-hybrid structures with other materials. This approach can be used for the development of new detection strategies as well as nanoelectronic structures and devices. Here we present a new method for the generation of unprecedented all-organic conjugated-polymer nanoparticle networks guided by DNA, based on a hierarchical self-assembly process. First, microphase separation of amphiphilic block copolymers induced the formation of spherical nanoobjects. As a second ordering concept, DNA base pairing has been employed for the controlled spatial definition of the conjugated-polymer particles within the bulk material. These networks offer the flexibility and the diversity of soft polymeric materials. Thus, simple chemical methodologies could be applied in order to tune the network's electrical, optical and mechanical properties. One- two- and three-dimensional networks have been successfully formed. Common to all morphologies is the integrity of the micelles consisting of DNA block copolymer (DBC), which creates an all-organic engineered network.

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences

PubMed Central

Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L.

2017-01-01

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. PMID:28628204

Efficient DNA binding and nuclear uptake by distamycin derivatives conjugated to octa-arginine sequences.

PubMed

Vázquez, Olalla; Blanco-Canosa, Juan B; Vázquez, M Eugenio; Martínez-Costas, Jose; Castedo, Luis; Mascareñas, José L

2008-11-24

Efficient targeting of DNA by designed molecules requires not only careful fine-tuning of their DNA-recognition properties, but also appropriate cell internalization of the compounds so that they can reach the cell nucleus in a short period of time. Previous observations in our group on the relatively high affinity displayed by conjugates between distamycin derivatives and bZIP basic regions for A-rich DNA sites, led us to investigate whether the covalent attachment of a positively charged cell-penetrating peptide to a distamycin-like tripyrrole might yield high affinity DNA binders with improved cell internalization properties. Our work has led to the discovery of synthetic tripyrrole-octa-arginine conjugates that are capable of targeting specific DNA sites that contain A-rich tracts with low nanomolar affinity; they simultaneously exhibit excellent membrane and nuclear translocation properties in living HeLa cells.

Quantitative Experimental Determination of Primer-Dimer Formation Risk by Free-Solution Conjugate Electrophoresis

PubMed Central

Desmarais, Samantha M.; Leitner, Thomas; Barron, Annelise E.

2012-01-01

DNA barcodes are short, unique ssDNA primers that “mark” individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis (FSCE) approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer-barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 basepairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive basepairs formed, yet non-consecutive basepairs did not create stable dimers even when 20 out of 30 possible basepairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation. PMID:22331820

Methods of DNA methylation detection

NASA Technical Reports Server (NTRS)

Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

2010-01-01

The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

Protein Detection via Direct Enzymatic Amplification of Short DNA Aptamers

PubMed Central

Fischer, Nicholas O.; Tarasow, Theodore M.; Tok, Jeffrey B.-H.

2008-01-01

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM). PMID:17980857

The key role of CYC2 during meiosis in Tetrahymena thermophila.

PubMed

Xu, Qianlan; Wang, Ruoyu; Ghanam, A R; Yan, Guanxiong; Miao, Wei; Song, Xiaoyuan

2016-04-01

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.

Interactions between inner membrane proteins in donor and recipient cells limit conjugal DNA transfer.

PubMed

Marrero, Joeli; Waldor, Matthew K

2005-06-01

Conjugation enables horizontal transmission of DNA among bacteria, thereby facilitating the rapid spread of genes such as those conferring resistance to antibiotics. Cell-cell contact is required for conjugative DNA transfer but does not ensure its success. The presence of certain plasmids in potential recipient cells inhibits redundant transfer of these plasmids from competent donors despite contact between donor and recipient cells. Here, we used two closely related integrating conjugative elements (ICEs), SXT and R391, to identify genes that inhibit redundant conjugative transfer. Cells containing SXT exclude transfer of a second copy of SXT but not R391 and vice versa. The specific exclusion of SXT and R391 is dependent upon variants of TraG and Eex, ICE-encoded inner membrane proteins in donor and recipient cells, respectively. We identified short sequences within each variant that determine the exquisite specificity of self-recognition; these data suggest that direct interactions between TraG and Eex mediate exclusion.

Quantum dots–DNA bioconjugates: synthesis to applications

PubMed Central

2016-01-01

Semiconductor nanoparticles particularly quantum dots (QDs) are interesting alternatives to organic fluorophores for a range of applications such as biosensing, imaging and therapeutics. Addition of a programmable scaffold such as DNA to QDs further expands the scope and applicability of these hybrid nanomaterials in biology. In this review, the most important stages of preparation of QD–DNA conjugates for specific applications in biology are discussed. Special emphasis is laid on (i) the most successful strategies to disperse QDs in aqueous media, (ii) the range of different conjugation with detailed discussion about specific merits and demerits in each case, and (iii) typical applications of these conjugates in the context of biology. PMID:27920898

Chemical conjugation of 2-hexadecynoic acid to C5-curcumin enhances its antibacterial activity against multi-drug resistant bacteria.

PubMed

Sanabria-Ríos, David J; Rivera-Torres, Yaritza; Rosario, Joshua; Gutierrez, Ricardo; Torres-García, Yeireliz; Montano, Nashbly; Ortíz-Soto, Gabriela; Ríos-Olivares, Eddy; Rodríguez, José W; Carballeira, Néstor M

2015-11-15

The first total synthesis of a C5-curcumin-2-hexadecynoic acid (C5-Curc-2-HDA, 6) conjugate was successfully performed. Through a three-step synthetic route, conjugate 6 was obtained in 13% overall yield and tested for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. Our results revealed that 6 was active against eight MRSA strains at MICs that range between 31.3 and 62.5 μg/mL. It was found that the presence of 2-hexadecynoic acid (2-HDA, 4) in conjugate 6 increased 4-8-fold its antibacterial activity against MRSA strains supporting our hypothesis that the chemical connection of 4 to C5-curcumin (2) increases the antibacterial activity of 2 against Gram-positive bacteria. Combinational index (CIn) values that range between 1.6 and 2.3 were obtained when eight MRSA strains were treated with an equimolar mixture of 2 and 4. These results demonstrated that an antagonistic effect is taking place. Finally, it was investigated whether conjugate 6 can affect the replication process of S. aureus, since this compound inhibited the supercoiling activity of the S. aureus DNA gyrase at minimum inhibitory concentrations (MIC) of 250 μg/mL (IC50=100.2±13.9 μg/mL). Moreover, it was observed that the presence of 4 in conjugate 6 improves the anti-topoisomerase activity of 2 towards S. aureus DNA gyrase, which is in agreement with results obtained from antibacterial susceptibility tests involving MRSA strains. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch.

PubMed

Hasegawa, Yusuke; Takada, Tadao; Nakamura, Mitsunobu; Yamana, Kazushige

2017-08-01

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch. Copyright © 2017 Elsevier Ltd. All rights reserved.

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nash, Rebekah Potts; Habibi, Sohrab; Cheng, Yuan

2010-11-15

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 {angstrom} crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that aremore » required for DNA nicking and religation were displaced up to 14 {angstrom} out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.« less

Inhibition of DNA nanotube-conjugated mTOR siRNA on the growth of pulmonary arterial smooth muscle cells.

PubMed

You, Zaichun; Qian, Hang; Wang, Changzheng; He, Binfeng; Yan, Jiawei; Mao, Chengde; Wang, Guansong

2015-12-01

Here we provide raw and processed data and methods behind mTOR siRNA loaded DNA nanotubes (siRNA-DNA-NTs) in the growth of pulmonary arterial smooth muscle cells (PASMCs) under both normoxic and hypoxic condition, and also related to (You et al., Biomaterials, 2015, 67:137-150, [1]). The MTT analysis, Semi-quantitative RT-PCR data presented here were used to probe cytotoxicity of mTOR siRNA-DNA-NT complex in its TAE-Mg(2+) buffer. siRNA-DNA-NTs have a lower cytotoxicity and higher transfection efficiency and can, based on inhibition of mTOR expression, decrease PASMCs growth both hypoxic and normal condition.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

PubMed

Bakhori, Noremylia Mohd; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-12-12

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10-9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

PubMed

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-12-01

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10(-9) M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

DNA-nanoparticle assemblies go organic: Macroscopic polymeric materials with nanosized features

PubMed Central

2012-01-01

Background One of the goals in the field of structural DNA nanotechnology is the use of DNA to build up 2- and 3-D nanostructures. The research in this field is motivated by the remarkable structural features of DNA as well as by its unique and reversible recognition properties. Nucleic acids can be used alone as the skeleton of a broad range of periodic nanopatterns and nanoobjects and in addition, DNA can serve as a linker or template to form DNA-hybrid structures with other materials. This approach can be used for the development of new detection strategies as well as nanoelectronic structures and devices. Method Here we present a new method for the generation of unprecedented all-organic conjugated-polymer nanoparticle networks guided by DNA, based on a hierarchical self-assembly process. First, microphase separation of amphiphilic block copolymers induced the formation of spherical nanoobjects. As a second ordering concept, DNA base pairing has been employed for the controlled spatial definition of the conjugated-polymer particles within the bulk material. These networks offer the flexibility and the diversity of soft polymeric materials. Thus, simple chemical methodologies could be applied in order to tune the network's electrical, optical and mechanical properties. Results and conclusions One- two- and three-dimensional networks have been successfully formed. Common to all morphologies is the integrity of the micelles consisting of DNA block copolymer (DBC), which creates an all-organic engineered network. PMID:22646980

Peptide-DNA conjugates as tailored bivalent binders of the oncoprotein c-Jun.

PubMed

Pazos, Elena; Portela, Cecilia; Penas, Cristina; Vázquez, M Eugenio; Mascareñas, José L

2015-05-21

We describe a ds-oligonucleotide-peptide conjugate that is able to efficiently dismount preformed DNA complexes of the bZIP regions of oncoproteins c-Fos and c-Jun (AP-1), and therefore might be useful as disrupters of AP-1-mediated gene expression pathways.

Chromosomal transfers in mycoplasmas: when minimal genomes go mobile.

PubMed

Dordet-Frisoni, Emilie; Sagné, Eveline; Baranowski, Eric; Breton, Marc; Nouvel, Laurent Xavier; Blanchard, Alain; Marenda, Marc Serge; Tardy, Florence; Sirand-Pugnet, Pascal; Citti, Christine

2014-11-25

Horizontal gene transfer (HGT) is a main driving force of bacterial evolution and innovation. This phenomenon was long thought to be marginal in mycoplasmas, a large group of self-replicating bacteria characterized by minute genomes as a result of successive gene losses during evolution. Recent comparative genomic analyses challenged this paradigm, but the occurrence of chromosomal exchanges had never been formally addressed in mycoplasmas. Here, we demonstrated the conjugal transfer of large chromosomal regions within and among ruminant mycoplasma species, with the incorporation of the incoming DNA occurring by homologous recombination into the recipient chromosome. By combining classical mating experiments with high-throughput next-generation sequencing, we documented the transfer of almost every position of the mycoplasma chromosome. Mycoplasma conjugation relies on the occurrence of an integrative conjugative element (ICE) in at least one parent cell. While ICE propagates horizontally from ICE-positive to ICE-negative cells, chromosomal transfers (CTs) occurred in the opposite direction, from ICE-negative to ICE-positive cells, independently of ICE movement. These findings challenged the classical mechanisms proposed for other bacteria in which conjugative CTs are driven by conjugative elements, bringing into the spotlight a new means for rapid mycoplasma innovation. Overall, they radically change our current views concerning the evolution of mycoplasmas, with particularly far-reaching implications given that over 50 species are human or animal pathogens. Horizontal gene transfers (HGT) shape bacterial genomes and are key contributors to microbial diversity and innovation. One main mechanism involves conjugation, a process that allows the simultaneous transfer of significant amounts of DNA upon cell-to-cell contact. Recognizing and deciphering conjugal mechanisms are thus essential in understanding the impact of gene flux on bacterial evolution. We addressed this issue in mycoplasmas, the smallest and simplest self-replicating bacteria. In these organisms, HGT was long thought to be marginal. We showed here that nearly every position of the Mycoplasma agalactiae chromosome could be transferred via conjugation, using an unconventional mechanism. The transfer involved DNA blocks containing up to 80 genes that were incorporated into the host chromosome by homologous recombination. These findings radically change our views concerning mycoplasma evolution and adaptation with particularly far-reaching implications given that over 50 species are human or animal pathogens. Copyright © 2014 Dordet-Frisoni et al.

Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

PubMed Central

Yamauchi, Y; Litwin, A; Adams, L; Zimmer, H; Hess, E V

1975-01-01

The antihypertensive drug hydralazine can induce in man a syndrome similar to spontaneous systemic lupus erythematosus (SLE). The pathogenesis of this drug-induced syndrome is not understood. In this investigation, five groups of rabbits were studied: group I, 10 rabbits hyperimmunized with hydralazine conjugated to human serum albumin (HSA) in complete Freund's adjuvant (CFA); group II, four rabbits with HSA in CFA; group III, four rabbits with CFA alone; group IV, five rabbits with hydralazine conjugated to rabbit serum albumin (RSA); and group V, four rabbits with a major metabolite of hydralazine conjugated to HSA. The rabbits immunized with hydralazine-HSA developed rising titers of antibodies to hydralazine and progressively increasing amounts of antibodies to both single-stranded and native DNA. The antibodies to DNA were cross-reactive with hydralazine as determined by inhibition of DNA binding and DNA hemagglutination tests. Similar results were obtained in rabbits immunized with the metabolite-HSA compound except the major hapten antibody response was to the metabolite. The DNA antibodies in this group were also capable of being absorbed by metabolite-HSA as well as hydralazine-HSA, indicative of the cross-reactivity between hydralazine and its metabolite. Immunization with hydralazine-RSA caused rabbits to produce antibodies to hydralazine but not to DNA, indicating the requirement for an immune response to the carrier protein in order for antibodies reactive with DNA to be produced. Thus, hyperimmunization of rabbits with hydralazine-protein conjugates may provide a useful animal model of SLE. The data suggests that an immune response to hydralazine may be important in human hydralazine-induced SLE. Images PMID:808562

Efficient self-assembly of DNA-functionalized fluorophores and gold nanoparticles with DNA functionalized silicon surfaces: the effect of oligomer spacers

PubMed Central

Milton, James A.; Patole, Samson; Yin, Huabing; Xiao, Qiang; Brown, Tom; Melvin, Tracy

2013-01-01

Although strategies for the immobilization of DNA oligonucleotides onto surfaces for bioanalytical and top-down bio-inspired nanobiofabrication approaches are well developed, the effect of introducing spacer molecules between the surface and the DNA oligonucleotide for the hybridization of nanoparticle–DNA conjugates has not been previously assessed in a quantitative manner. The hybridization efficiency of DNA oligonucleotides end-labelled with gold nanoparticles (1.4 or 10 nm diameter) with DNA sequences conjugated to silicon surfaces via hexaethylene glycol phosphate diester oligomer spacers (0, 1, 2, 6 oligomers) was found to be independent of spacer length. To quantify both the density of DNA strands attached to the surfaces and hybridization with the surface-attached DNA, new methodologies have been developed. Firstly, a simple approach based on fluorescence has been developed for determination of the immobilization density of DNA oligonucleotides. Secondly, an approach using mass spectrometry has been created to establish (i) the mean number of DNA oligonucleotides attached to the gold nanoparticles and (ii) the hybridization density of nanoparticle–oligonucleotide conjugates with the silicon surface–attached complementary sequence. These methods and results will be useful for application with nanosensors, the self-assembly of nanoelectronic devices and the attachment of nanoparticles to biomolecules for single-molecule biophysical studies. PMID:23361467

Interleukin-12 plasmid DNA delivery using l-thyroxine-conjugated polyethylenimine nanocarriers

NASA Astrophysics Data System (ADS)

Dehshahri, Ali; Sadeghpour, Hossein; Kazemi Oskuee, Reza; Fadaei, Mahin; Sabahi, Zahra; Alhashemi, Samira Hossaini; Mohazabieh, Erfaneh

2014-05-01

In this study, l-thyroxine was covalently grafted on 25 kDa branched polyethylenimine (PEI), and the ability of the nano-sized polyplexes for transferring plasmid encoding interleukin-12 (IL-12) gene was evaluated. As there are several problems in systemic administration of recombinant IL-12 protein, local expression of the plasmid encoding IL-12 gene inside the tumor tissue has been considered as an effective alternative approach. The l-thyroxine-conjugated PEI polyplexes were prepared using pUMVC3-hIL12 plasmid, and their transfection activity was determined in HepG2 human liver carcinoma and Neuro2A neuroblastoma cell lines. The polyplexes characterized in terms of DNA condensation ability, particle size, zeta potential, and buffering capacity as well as cytotoxicity and resistance to enzyme digestion. The results revealed that l-thyroxine conjugation of PEI increased gene transfer ability by up to two fold relative to unmodified 25 kDa PEI, the gold standard for non-viral gene delivery, with the highest increase occurring at degrees of conjugation around 10 %. pDNA condensation tests and dynamic light scattering measurements exhibited the ability of PEI conjugates to optimally condense the plasmid DNA into polyplexes in the size range around 200 nm. The modified polymers showed remarkable buffering capacity and protection against enzymatic degradation comparable to that of unmodified PEI. These results suggest that l-thyroxine conjugation of PEI is a simple modification strategy for future investigations aimed at developing a targeting gene vehicle.

Site-Directed Photoproteolysis of 8-Oxoguanine DNA Glycosylase 1 (OGG1) by Specific Porphyrin-Protein Probe Conjugates: A Strategy to Improve the Effectiveness of Photodynamic Therapy for Cancer

PubMed Central

Conlon, Kimberly A.; Berrios, Miguel

2007-01-01

The specific light-induced, non-enzymatic photolysis of mOGG1 by porphyrin-conjugated or rose bengal-conjugated streptavidin and porphyrin-conjugated or rose bengal-conjugated first specific or secondary anti-IgG antibodies is reported. The porphyrin chlorin e6 and rose bengal were conjugated to either streptavidin, rabbit anti-mOGG1 primary specific antibody fractions or goat anti-rabbit IgG secondary antibody fractions. Under our experimental conditions, visible light of wavelengths greater than 600 nm induced the non-enzymatic degradation of mOGG1 when this DNA repair enzyme either directly formed a complex with chlorin e6-conjugated anti-mOGG1 primary specific antibodies or indirectly formed complexes with either streptavidin-chlorin e6 conjugates and biotinylated first specific anti-mOGG1 antibodies or first specific anti-mOGG1antibodies and chlorin e6-conjugated anti-rabbit IgG secondary antibodies. Similar results were obtained when rose bengal was used as photosensitizer instead of chlorine e6. The rate of the photochemical reaction of mOGG1 site-directed by all three chlorine e6 antibody complexes was not affected by the presence of the singlet oxygen scavenger sodium azide. Site-directed photoactivatable probes having the capacity to generate reactive oxygen species (ROS) while destroying the DNA repair system in malignant cells and tumors may represent a powerful strategy to boost selectivity, penetration and efficacy of current photodynamic (PDT) therapy methodologies. PMID:17251034

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

PubMed

Gill, Pooria; Forouzandeh, Mehdi; Rahbarizadeh, Fatemeh; Ramezani, Reihaneh; Rasaee, Mohammad Javad

2006-01-01

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Prokaryotic Information Games: How and When to Take up and Secrete DNA.

PubMed

Stingl, Kerstin; Koraimann, Günther

2017-01-01

Besides transduction via bacteriophages natural transformation and bacterial conjugation are the most important mechanisms driving bacterial evolution and horizontal gene spread. Conjugation systems have evolved in eubacteria and archaea. In Gram-positive and Gram-negative bacteria, cell-to-cell DNA transport is typically facilitated by a type IV secretion system (T4SS). T4SSs also mediate uptake of free DNA in Helicobacter pylori, while most transformable bacteria use a type II secretion/type IV pilus system. In this chapter, we focus on how and when bacteria "decide" that such a DNA transport apparatus is to be expressed and assembled in a cell that becomes competent. Development of DNA uptake competence and DNA transfer competence is driven by a variety of stimuli and often involves intricate regulatory networks leading to dramatic changes in gene expression patterns and bacterial physiology. In both cases, genetically homogeneous populations generate a distinct subpopulation that is competent for DNA uptake or DNA transfer or might uniformly switch into competent state. Phenotypic conversion from one state to the other can rely on bistable genetic networks that are activated stochastically with the integration of external signaling molecules. In addition, we discuss principles of DNA uptake processes in naturally transformable bacteria and intend to understand the exceptional use of a T4SS for DNA import in the gastric pathogen H. pylori. Realizing the events that trigger developmental transformation into competence within a bacterial population will eventually help to create novel and effective therapies against the transmission of antibiotic resistances among pathogens.

Water-Soluble Conjugated Polymers: Self-Assembly and Biosensor Applications

NASA Astrophysics Data System (ADS)

Bazan, Guillermo

2005-03-01

Homogeneous assays can be designed which take advantage of the optical amplification of conjugated polymers and the self-assembly characteristic of aqueous polyelectrolytes. For example, a ssDNA sequence sensor comprises an aqueous solution containing a cationic water soluble conjugated polymer such as poly(9,9-bis(trimethylammonium)-hexyl)-fluorene phenylene) with a peptide nucleic acid (PNA) labeled with a dye (PNA-C*). Signal transduction is controlled by hybridization of the neutral PNA-C* probe and the negative ssDNA target, resulting in favorable electrostatic interactions between the hybrid complex and the cationic polymer. Distance requirements for Förster energy transfer are thus met only when ssDNA of complementary sequence to the PNA-C* probe is present. Signal amplification by the conjugated polymer provides fluorescein emission >25 times higher than that of the directly excited dye. Transduction by electrostatic interactions followed by energy transfer is a general strategy. Examples involving other biomolecular recognition events, such as DNA/DNA, RNA/protein and RNA/RNA, will also be provided. The mechanism of biosensing will be discussed, with special attention to the varying contributions of hydrophobic and electrostatic forces, polymer conformation, charge density, local concentration of C*s and tailored defect sites for aggregation-induced optical changes. Finally, the water solubility of these conjugated polymers opens possibilities for spin casting onto organic materials, without dissolving the underlying layers. This property is useful for fabricating multilayer organic optoelectronic devices by simple solution techniques.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

PubMed Central

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-01-01

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense. PMID:25587406

How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

PubMed

Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

2017-03-15

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Light-controlled cellular internalization and cytotoxicity of nucleic acid-binding agents. Studies in vitro and in zebrafish embryos

PubMed Central

Penas, Cristina; Sánchez, Mateo I.; Guerra-Varela, Jorge; Sanchez-Piñón, Laura; Vázquez, M. Eugenio; Mascareñas, José L.

2016-01-01

We have synthesized oligoarginine conjugates of selected DNA-binding agents (a bisbenzamidine, acridine and thiazole orange) and demonstrated that the DNA binding and cell internalization properties of such conjugates can be inhibited by appending a negatively charged oligoglutamic tail through a photolabile linker. Irradiation with UV light releases the parent octaarginine conjugates, thus restoring their cell internalization and biological activity. Preliminary assays using zebrafish embryos demonstrates the potential of this prodrug strategy for controlling in vivo cytotoxicity. PMID:26534774

Specific Inhibition of the transcription factor Ci by a Cobalt(III)-Schiff base-DNA conjugate

PubMed Central

Hurtado, Ryan R.; Harney, Allison S.; Heffern, Marie C.; Holbrook, Robert J.; Holmgren, Robert A.; Meade, Thomas J.

2012-01-01

We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas. It is well known that Hh regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. In Drosophila the function of the Gli proteins is performed by a single transcription factor with an identical DNA binding consensus sequence, Cubitus Interruptus (Ci). We have demonstrated previously that conjugation of a specific 17 base-pair oligonucleotide to a Co(III) Schiff base complex results in a targeted inhibitor of the Snail family C2H2 zinc finger transcription factors. Modification of the oligonucleotide sequence in the Co(III) Schiff base-DNA conjugate to that of Ci’s consensus sequence (Co(III)-Ci) generates an equally selective inhibitor of Ci. Co(III)-Ci irreversibly binds the Ci zinc finger domain and prevents it from binding DNA in vitro. In a Ci responsive tissue culture reporter gene assay, Co(III)-Ci reduces the transcriptional activity of Ci in a concentration dependent manner. In addition, injection of wild-type Drosophila embryos with Co(III)-Ci phenocopies a Ci loss of function phenotype, demonstrating effectiveness in vivo. This study provides evidence that Co(III) Schiff base-DNA conjugates are a versatile class of specific and potent tools for studying zinc finger domain proteins and have potential applications as customizable anti-cancer therapeutics. PMID:22214326

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

PubMed Central

Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

2016-01-01

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

One-to-one quantum dot-labeled single long DNA probes.

PubMed

He, Shibin; Huang, Bi-Hai; Tan, Junjun; Luo, Qing-Ying; Lin, Yi; Li, Jun; Hu, Yong; Zhang, Lu; Yan, Shihan; Zhang, Qi; Pang, Dai-Wen; Li, Lijia

2011-08-01

Quantum dots (QDs) have been received most attention due to their unique properties. Constructing QDs conjugated with certain number of biomolecules is considered as one of the most important research goals in nanobiotechnology. In this study, we report polymerase chain reaction (PCR) amplification of primer oligonucleotides bound to QDs, termed as QD-based PCR. Characterization of QD-based PCR products by gel electrophoresis and atomic force microscopy showed that QD-labeled long DNA strands were synthesized and only a single long DNA strand was conjugated with a QD. The QD-based PCR products still kept fluorescence properties. Moreover, the one-to-one QD-labeled long DNA conjugates as probes could detect a single-copy gene on maize chromosomes by fluorescence in situ hybridization. Labeling a single QD to a single long DNA will make detection of small single-copy DNA fragments, quantitative detection and single molecule imaging come true by nanotechnology, and it will promote medical diagnosis and basic biological research as well as nano-material fabrication. Copyright © 2011 Elsevier Ltd. All rights reserved.

Dielectrophoresis of gold nanoparticles conjugated to DNA origami structures

PubMed Central

Wiens, Matthew; Lakatos, Mathias; Heerwig, Andreas; Ostermaier, Frieder; Haufe, Nora

2016-01-01

Summary DNA nanostructures are promising construction materials to bridge the gap between self-assembly of functional molecules and conventional top-down fabrication methods in nanotechnology. Their positioning onto specific locations of a microstructured substrate is an important task towards this aim. Here we study manipulation and positioning of pristine and of gold nanoparticle-conjugated tubular DNA origami structures using ac dielectrophoresis. The dielectrophoretic behavior was investigated employing fluorescence microscopy. For the pristine origami, a significant dielectrophoretic response was found to take place in the megahertz range, whereas, due to the higher polarizability of the metallic nanoparticles, the nanoparticle/DNA hybrid structures required a lower electrical field strength and frequency for a comparable trapping at the edges of the electrode structure. The nanoparticle conjugation additionally resulted in a remarkable alteration of the DNA structure arrangement. The growth of linear, chain-like structures in between electrodes at applied frequencies in the megahertz range was observed. The long-range chain formation is caused by a local, gold nanoparticle-induced field concentration along the DNA nanostructures, which in turn, creates dielectrophoretic forces that enable the observed self-alignment of the hybrid structures. PMID:27547612

What Controls the "Off/On Switch" in the Toehold-Mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles?

PubMed

Yao, Dongbao; Wang, Bei; Xiao, Shiyan; Song, Tingjie; Huang, Fujian; Liang, Haojun

2015-06-30

In DNA dynamic nanotechnology, a toehold-mediated DNA strand-displacement reaction has demonstrated its capability in building complex autonomous system. In most cases, the reaction is performed in pure DNA solution that is essentially a one-phase system. In the present work, we systematically investigated the reaction in a heterogeneous media, in which the strand that implements a displacing action is conjugated on gold nanoparticles. By monitoring the kinetics of spherical nucleic acid (SNA) assembly driven by toehold-mediated strand displacement reaction, we observed significant differences, i.e., the abrupt jump in behavior of an "off/on switch", in the reaction rate when the invading toehold was extended to eight bases from seven bases. These phenomena are attributed to the effect of steric hindrance arising from the high density of invading strand conjugated to AuNPs. Based on these studies, an INHIBIT logic gate presenting good selectivity was developed.

Incorporation of native antibodies and Fc-fusion proteins on DNA nanostructures via a modular conjugation strategy† †Electronic supplementary information (ESI) available: Experimental methods, DNA origami design, DNA sequences, and additional experimental data. See DOI: 10.1039/c7cc04178k

PubMed Central

Rosier, Bas J. H. M.; Cremers, Glenn A. O.; Engelen, Wouter; Merkx, Maarten; Brunsveld, Luc

2017-01-01

A photocrosslinkable protein G variant was used as an adapter protein to covalently and site-specifically conjugate an antibody and an Fc-fusion protein to an oligonucleotide. This modular approach enables straightforward decoration of DNA nanostructures with complex native proteins while retaining their innate binding affinity, allowing precise control over the nanoscale spatial organization of such proteins for in vitro and in vivo biomedical applications. PMID:28617516

Optically degradable dendrons for temporary adhesion of proteins to DNA.

PubMed

Kostiainen, Mauri A; Kotimaa, Juha; Laukkanen, Marja-Leena; Pavan, Giovanni M

2010-06-18

Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N-maleimide-cored polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4-conjugate addition to give one-to-one protein-polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single-chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein-dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (lambda approximately 350 nm) can cleave the photolabile o-nitrobenzyl-linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

PubMed

Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

2015-08-25

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

One-Dimensional Multichromophor Arrays Based on DNA: From Self-Assembly to Light-Harvesting.

PubMed

Ensslen, Philipp; Wagenknecht, Hans-Achim

2015-10-20

Light-harvesting complexes collect light energy and deliver it by a cascade of energy and electron transfer processes to the reaction center where charge separation leads to storage as chemical energy. The design of artificial light-harvesting assemblies faces enormous challenges because several antenna chromophores need to be kept in close proximity but self-quenching needs to be avoided. Double stranded DNA as a supramolecular scaffold plays a promising role due to its characteristic structural properties. Automated DNA synthesis allows incorporation of artificial chromophore-modified building blocks, and sequence design allows precise control of the distances and orientations between the chromophores. The helical twist between the chromophores, which is induced by the DNA framework, controls energy and electron transfer and thereby reduces the self-quenching that is typically observed in chromophore aggregates. This Account summarizes covalently multichromophore-modified DNA and describes how such multichromophore arrays were achieved by Watson-Crick-specific and DNA-templated self-assembly. The covalent DNA systems were prepared by incorporation of chromophores as DNA base substitutions (either as C-nucleosides or with acyclic linkers as substitutes for the 2'-deoxyribofuranoside) and as DNA base modifications. Studies with DNA base substitutions revealed that distances but more importantly relative orientations of the chromophores govern the energy transfer efficiencies and thereby the light-harvesting properties. With DNA base substitutions, duplex stabilization was faced and could be overcome, for instance, by zipper-like placement of the chromophores in both strands. For both principal structural approaches, DNA-based light-harvesting antenna could be realized. The major disadvantages, however, for covalent multichromophore DNA conjugates are the poor yields of synthesis and the solubility issues for oligonucleotides with more than 5-10 chromophore modifications in a row. A logical alternative approach is to leave out the phosphodiester bridges between the chromophores and let chromophore-nucleoside conjugates self-assemble specifically along single stranded DNA as template. The self-organization of chromophores along the DNA template based on canonical base pairing would be advantageous because sequence selective base pairing could provide a structural basis for programmed complexity within the chromophore assembly. The self-assembly is governed by two interactions. The chromophore-nucleoside conjugates as guest molecules are recognized via hydrogen bonds to the corresponding counter bases in the single stranded DNA template. Moreover, the π-π interactions between the stacked chromophores stabilize these self-assembled constructs with increasing length. Longer DNA templates are more attractive for self-assembled antenna. The helicity in the stack of porphyrins as guest molecules assembled on the DNA template can be switched by environmental changes, such as pH variations. DNA-templated stacks of ethynyl pyrene and nile red exhibit left-handed chirality, which stands in contrast to similar covalent multichromophore-DNA conjugates with enforced right-handed helicity. With ethynyl nile red, it is possible to occupy every available binding site on the templates. Mixed assemblies of ethynyl pyrene and nile red show energy transfer and thereby provide a proof-of-principle that simple light-harvesting antennae can be obtained in a noncovalent and self-assembled fashion. With respect to the next important step, chemical storage of the absorbed light energy, future research has to focus on the coupling of sophisticated DNA-based light-harvesting antenna to reaction centers.

Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

NASA Astrophysics Data System (ADS)

Krüger, Harald R.; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

2015-02-01

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening. Electronic supplementary information (ESI) available: Including detailed synthetic procedures of the dye and conjugate synthesis, as well as cellular uptake and inhibitor studies. See DOI: 10.1039/c4nr04467c

The synthesis and biological evaluation of new DNA-directed alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates containing a urea linker.

PubMed

Marvania, Bhavin; Kakadiya, Rajesh; Christian, Wilson; Chen, Tai-Lin; Wu, Ming-Hsi; Suman, Sharda; Tala, Kiran; Lee, Te-Chang; Shah, Anamik; Su, Tsann-Long

2014-08-18

We synthesized a series of phenyl N-mustard-4-anilinoquinoline conjugates to study their antitumorigenic effects. These agents were prepared by the condensation of 4-[N,N-bis(2-chloroethyl)amino]phenyl isocyanate with 6-amino-4-methylamino or 4-anilinoquinolines. The structure-activity relationship (SAR) studies revealed that the C2-methylquinoline derivatives (18a-o) were generally more cytotoxic than the C2-phenylquinoline conjugates (23a-d) in inhibiting the cell growth of various human tumor cell lines in vitro. However, the methylamino or aniline substituents at C4 of quinoline did not influence the cytotoxic effects. The title conjugates were capable of inducing DNA cross-linking and promoting cell-cycle arrest at the G2/M phase. This study demonstrates that phenyl N-mustard-4-anilinoquinoline conjugates are generally more potent than phenyl N-mustard-4-anilinoquinazoline conjugates against the cell growth of various tumor cell-lines. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

DNA unwinding by Escherichia coli DNA helicase I (TraI) provides evidence for a processive monomeric molecular motor.

PubMed

Sikora, Bartek; Eoff, Robert L; Matson, Steven W; Raney, Kevin D

2006-11-24

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

Sequence-selective binding of C8-conjugated pyrrolobenzodiazepines (PBDs) to DNA.

PubMed

Basher, Mohammad A; Rahman, Khondaker Miraz; Jackson, Paul J M; Thurston, David E; Fox, Keith R

2017-11-01

DNA footprinting and melting experiments have been used to examine the sequence-specific binding of C8-conjugates of pyrrolobenzodiazepines (PBDs) and benzofused rings including benzothiophene and benzofuran, which are attached using pyrrole- or imidazole-containing linkers. The conjugates modulate the covalent attachment points of the PBDs, so that they bind best to guanines flanked by A/T-rich sequences on either the 5'- or 3'-side. The linker affects the binding, and pyrrole produces larger changes than imidazole. Melting studies with 14-mer oligonucleotide duplexes confirm covalent attachment of the conjugates, which show a different selectivity to anthramycin and reveal that more than one ligand molecule can bind to each duplex. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

PubMed Central

Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

2016-01-01

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

Free-solution electrophoretic separations of DNA–drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength

PubMed Central

Albrecht, Jennifer Coyne; Kerby, Matthew B.; Niedringhaus, Thomas P.; Lin, Jennifer S.; Wang, Xiaoxiao; Barron, Annelise E.

2012-01-01

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of Free-Solution Conjugate Electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. Single-stranded DNA separations in “gels” have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE’s ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with pHEA-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags. PMID:21500207

Synthesis, DNA binding ability and anticancer activity of 2-heteroaryl substituted benzimidazoles linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates.

PubMed

Kamal, Ahmed; Pogula, Praveen Kumar; Khan, Mohammed Naseer Ahmed; Seshadri, Bobburi Naga; Sreekanth, Kokkonda

2013-08-01

As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential anticancer agents, a series of heteroaryl substituted benzimidazole linked PBD conjugates has been synthesized and evaluated for their anticancer potential in 60 human cancer cell lines. Most of the compounds exhibited promising anticancer activity and interestingly, compounds 4c and 4d displayed significant activity in most of the cell lines tested. Whereas, compound 4e showed selectivity in renal cancer cells with GI50 values of <10 and 70 nM against RXF 393 and UO-31 cell lines, respectively. Further, these compounds also showed significant DNA-binding affinity by thermal denaturation study using duplex form of calf thymus (CT) DNA.

Self-assembly of nucleic acids, silk and hybrid materials thereof.

PubMed

Humenik, Martin; Scheibel, Thomas

2014-12-17

Top-down approaches based on etching techniques have almost reached their limits in terms of dimension. Therefore, novel assembly strategies and types of nanomaterials are required to allow technological advances. Self-assembly processes independent of external energy sources and unlimited in dimensional scaling have become a very promising approach. Here,we highlight recent developments in self-assembled DNA-polymer, silk-polymer and silk-DNA hybrids as promising materials with biotic and abiotic moieties for constructing complex hierarchical materials in ‘bottom-up’ approaches. DNA block copolymers assemble into nanostructures typically exposing a DNA corona which allows functionalization, labeling and higher levels of organization due to its specific addressable recognition properties. In contrast, self-assembly of natural silk proteins as well as their recombinant variants yields mechanically stable β-sheet rich nanostructures. The combination of silk with abiotic polymers gains hybrid materials with new functionalities. Together, the precision of DNA hybridization and robustness of silk fibrillar structures combine in novel conjugates enable processing of higher-order structures with nanoscale architecture and programmable functions.

Self-assembly of nucleic acids, silk and hybrid materials thereof

NASA Astrophysics Data System (ADS)

Humenik, Martin; Scheibel, Thomas

2014-12-01

Top-down approaches based on etching techniques have almost reached their limits in terms of dimension. Therefore, novel assembly strategies and types of nanomaterials are required to allow technological advances. Self-assembly processes independent of external energy sources and unlimited in dimensional scaling have become a very promising approach. Here, we highlight recent developments in self-assembled DNA-polymer, silk-polymer and silk-DNA hybrids as promising materials with biotic and abiotic moieties for constructing complex hierarchical materials in ‘bottom-up’ approaches. DNA block copolymers assemble into nanostructures typically exposing a DNA corona which allows functionalization, labeling and higher levels of organization due to its specific addressable recognition properties. In contrast, self-assembly of natural silk proteins as well as their recombinant variants yields mechanically stable β-sheet rich nanostructures. The combination of silk with abiotic polymers gains hybrid materials with new functionalities. Together, the precision of DNA hybridization and robustness of silk fibrillar structures combine in novel conjugates enable processing of higher-order structures with nanoscale architecture and programmable functions.

A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity.

PubMed

Miller, Michael L; Fishkin, Nathan E; Li, Wei; Whiteman, Kathleen R; Kovtun, Yelena; Reid, Emily E; Archer, Katie E; Maloney, Erin K; Audette, Charlene A; Mayo, Michele F; Wilhelm, Alan; Modafferi, Holly A; Singh, Rajeeva; Pinkas, Jan; Goldmacher, Victor; Lambert, John M; Chari, Ravi V J

2016-08-01

The promise of tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. ©2016 AACR. ©2016 American Association for Cancer Research.

Optical properties of DNA induced starch capped PbS, CdS and PbS/CdS nanocomposites

NASA Astrophysics Data System (ADS)

Das, D.; Konwar, R.; Kalita, P. K.

2015-08-01

Starch capped PbS, CdS and PbS-CdS nanocomposites are conjugated with Calf-Thymus DNA. All the materials are characterized by X-ray diffraction, high-resolution transmission electron microscopy, UV-Vis spectroscopy and photoluminescence spectroscopy. The x-ray diffraction patterns of PbS and CdS show that the materials possess polycrystalline having both cubic and hexagonal phases. High resolution transmission electron microscopic results (HRTEM) shows PbS nanoparticles of size 3 nm and that of CdS nanoparticles having average size 4 nm which exhibit tendency of agglomeration. In case of PbS/CdS, it exhibits different types of nanosheets. The UV absorption spectra of all the samples exhibit clear blue-shift with the respective bulk absorption edges. This is attributed to the strong quantum confinement in the materials. The absorption spectra also exhibit increase of the band gaps from 2.25 to 4.35 eV for PbS; 2.25-4.2 eV for CdS with decrease of molarities from 0.1 to 0.001 M as well as conjugated with DNA. The photoluminescence spectra of all PbS, CdS and PbS/CdS composites synthesized at 0.1 M molar concentration show a further blue shift and an enhancement of intensity after conjugation with DNA, but the effect is reversed i.e. occurrence of red shift and reduction of intensity for those having 0.01 M. This is due to the two competing processes of surface passivation as well as stabilization of nanocomposites governed by bio-molecules and that of Dexter energy transfer with the effective charge separation. The result shows the applicability of the materials in development of biological labels and biosensors.

Nano-encrypted Morse code: a versatile approach to programmable and reversible nanoscale assembly and disassembly.

PubMed

Wong, Ngo Yin; Xing, Hang; Tan, Li Huey; Lu, Yi

2013-02-27

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code "NANO" and reversible exchange of uppercase letter "I" with lowercase "i". The yields of the conjugations are high (>90%), and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates.

Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction.

PubMed

Xu, Xue-tao; Liang, Kai-yi; Zeng, Jia-ying

2015-02-15

A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA-invertase conjugation on the surface of Streptavidin-MNBs. The DNA-invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000 fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of Polymer Conjugation on Hybridization Thermodynamics of Oligonucleic Acids.

PubMed

Ghobadi, Ahmadreza F; Jayaraman, Arthi

2016-09-15

In this work, we perform coarse-grained (CG) and atomistic simulations to study the effects of polymer conjugation on hybridization/melting thermodynamics of oligonucleic acids (ONAs). We present coarse-grained Langevin molecular dynamics simulations (CG-NVT) to assess the effects of the polymer flexibility, length, and architecture on hybridization/melting of ONAs with different ONA duplex sequences, backbone chemistry, and duplex concentration. In these CG-NVT simulations, we use our recently developed CG model of ONAs in implicit solvent, and treat the conjugated polymer as a CG chain with purely repulsive Weeks-Chandler-Andersen interactions with all other species in the system. We find that 8-100-mer linear polymer conjugation destabilizes 8-mer ONA duplexes with weaker Watson-Crick hydrogen bonding (WC H-bonding) interactions at low duplex concentrations, while the same polymer conjugation has an insignificant impact on 8-mer ONA duplexes with stronger WC H-bonding. To ensure the configurational space is sampled properly in the CG-NVT simulations, we also perform CG well-tempered metadynamics simulations (CG-NVT-MetaD) and analyze the free energy landscape of ONA hybridization for a select few systems. We demonstrate that CG-NVT-MetaD simulation results are consistent with the CG-NVT simulations for the studied systems. To examine the limitations of coarse-graining in capturing ONA-polymer interactions, we perform atomistic parallel tempering metadynamics simulations at well-tempered ensemble (AA-MetaD) for a 4-mer DNA in explicit water with and without conjugation to 8-mer poly(ethylene glycol) (PEG). AA-MetaD simulations also show that, for a short DNA duplex at T = 300 K, a condition where the DNA duplex is unstable, conjugation with PEG further destabilizes DNA duplex. We conclude with a comparison of results from these three different types of simulations and discuss their limitations and strengths.

Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

PubMed

Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

2016-09-10

Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

PubMed

Edelson, Benjamin S; Best, Timothy P; Olenyuk, Bogdan; Nickols, Nicholas G; Doss, Raymond M; Foister, Shane; Heckel, Alexander; Dervan, Peter B

2004-01-01

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

NASA Astrophysics Data System (ADS)

Lin, Wen Jen; Chien, Wei Hsuan

2015-09-01

The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly( d,l-lactide- co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal

PubMed Central

2009-01-01

The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses indicate that the DNA interstand cross-links contain a mixture of carbinolamine and Schiff base, with the carbinolamine forms of the linkages predominating in duplex DNA. The reduced derivatives of the enal-mediated N2-dG:N2-dG interstrand cross-links can be processed in mammalian cells by a mechanism not requiring homologous recombination. Mutations are rarely generated during processing of these cross-links. In contrast, the reduced acrolein-mediated N2-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol κ in human, are implicated in error-free bypass of model acrolein-mediated N2-dG secondary adducts, the interstrand cross-links, and the peptide conjugates. PMID:19397281

Enhancing Cell Nucleus Accumulation and DNA Cleavage Activity of Anti-Cancer Drug via Graphene Quantum Dots

NASA Astrophysics Data System (ADS)

Wang, Chong; Wu, Congyu; Zhou, Xuejiao; Han, Ting; Xin, Xiaozhen; Wu, Jiaying; Zhang, Jingyan; Guo, Shouwu

2013-10-01

Graphene quantum dots (GQDs) maintain the intrinsic layered structural motif of graphene but with smaller lateral size and abundant periphery carboxylic groups, and are more compatible with biological system, thus are promising nanomaterials for therapeutic applications. Here we show that GQDs have a superb ability in drug delivery and anti-cancer activity boost without any pre-modification due to their unique structural properties. They could efficiently deliver doxorubicin (DOX) to the nucleus through DOX/GQD conjugates, because the conjugates assume different cellular and nuclear internalization pathways comparing to free DOX. Also, the conjugates could enhance DNA cleavage activity of DOX markedly. This enhancement combining with efficient nuclear delivery improved cytotoxicity of DOX dramatically. Furthermore, the DOX/GQD conjugates could also increase the nuclear uptake and cytotoxicity of DOX to drug-resistant cancer cells indicating that the conjugates may be capable to increase chemotherapy efficacy of anti-cancer drugs that are suboptimal due to the drug resistance.

A common anchor facilitated GO-DNA nano-system for multiplex microRNA analysis in live cells.

PubMed

Yu, Jiantao; He, Sihui; Shao, Chen; Zhao, Haoran; Li, Jing; Tian, Leilei

2018-04-19

The design of a nano-system for the detection of intracellular microRNAs is challenging as it must fulfill complex requirements, i.e., it must have a high sensitivity to determine the dynamic expression level, a good reliability for multiplex and simultaneous detection, and a satisfactory biostability to work in biological environments. Instead of employing a commonly used physisorption or a full-conjugation strategy, here, a GO-DNA nano-system was developed under graft/base-pairing construction. The common anchor sequence was chemically grafted to GO to base-pair with various microRNA probes; and the hybridization with miRNAs drives the dyes on the probes to leave away from GO, resulting in "turned-on" fluorescence. This strategy not only simplifies the synthesis but also efficiently balances the loading yields of different probes. Moreover, the conjugation yield of GO with a base-paired hybrid has been improved by more than two-fold compared to that of the conjugation with a single strand. We demonstrated that base-paired DNA probes could be efficiently delivered into cells along with GO and are properly stabilized by the conjugated anchor sequence. The resultant GO-DNA nano-system exhibited high stability in a complex biological environment and good resistance to nucleases, and was able to accurately discriminate various miRNAs without cross-reaction. With all of these positive features, the GO-DNA nano-system can simultaneously detect three miRNAs and monitor their dynamic expression levels.

Rapid aptasensor capable of simply diagnosing prostate cancer.

PubMed

Cha, Timothy; Cho, Sandy; Kim, Young Teck; Lee, Ji Hoon

2014-12-15

Using guanine (G)-rich DNA aptamer-conjugated 6-carboxyfluorescein (6-FAM) capable of rapidly capturing prostate specific antigen (PSA) in human serum, cost-effective and simple biosensor with guanine chemiluminescence detection was developed for early diagnosis of prostate cancer. Free G-rich DNA aptamer-conjugated 6-FAM emits bright light in guanine chemiluminescence reaction based on the principle of chemiluminescent resonance energy transfer (CRET). However, G-rich DNA aptamer-conjugated 6-FAM bound with PSA cannot emit light because PSA acts as a strong interference in CRET between 6-FAM and high-energy intermediate formed from the reaction of 3,4,5-trimethoxylphenylglyoxal (TMPG) and guanine of G-rich DNA aptamer. A chemiluminescent biosensor, developed using the different properties of G-rich DNA aptamer-conjugated 6-FAM in the absence and presence of PSA in guanine chemiluminescence reaction, was able to quantify trace levels of PSA in human serum within 30 min without time-consuming and complicated procedures (e.g., multiple incubation and washings) required for conventional immunoassays operated with expensive and intractable antibodies. The limit of detection of chemiluminescent biosensor having a wide linear dynamic range (1.9-125 ng/ml) was 1.0 ng/ml. The excellent correlation (R=0.985) between chemiluminescent biosensor and conventional enzyme immunoassay indicates that the accurate, precise, and rapid chemiluminescent biosensor can be applied as a new method for early diagnosis of prostate cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Application of quantum-dots for analysis of nanosystems by either utilizing or preventing FRET

NASA Astrophysics Data System (ADS)

Kim, Joong H.; Chaudhary, Sumit; Stephens, Jared P.; Singh, Krishna V.; Ozkan, Mihrimah

2005-04-01

We have developed conjugates with quantum-dots (QDs) for the purpose of analysis of nanosystems that are organic or inorganic in nature such as DNA and carbon nanotubes. First, by employing Florescence Resonant Energy Transfer (FRET) principles, a hybrid molecular beacon conjugates are synthesized. For water- solubilization of QDs, we modified the surface of CdSe-ZnS core-shell QD by using mercaptoacetic acid ligand. This modification does not affect the size of QDs from that of unmodified QDs. After linking molecular beacons to the carboxyl groups of the modified QDs using 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, hybrid molecular beacons are prepared as a DNA probe. After hybridization with specific target DNA and non-specific target DNA, the hybrid conjugates show high specificity to the target DNA with 5-fold increase in the intensity of fluorescence. By developing atomic model of the conjugates, we calculated with 8 numbers of molecular beacons on a single quantum dots, we could increase the efficiency of FRET up to 90%. In other hands, for application of quantum dots to the carbon nanotubes, FRET is a barrier. Thus, after employing 1 % sodium-dodecyl-sulfonate (SDS), single-walled carbon nanotubes are decorated with QDs at their outer surface. This enables fluorescent microscopy imaging of single-walled carbon nanotubes which is a more common technique than electron microscopy. In summary, QDs can be used for analysis or detection of both organic and inorganic based nanosystems.

Multi-pedal DNA walker biosensors based on catalyzed hairpin assembly and isothermal strand-displacement polymerase reaction for the chemiluminescent detection of proteins.

PubMed

Li, Ningxing; Du, Mingyuan; Liu, Yucheng; Ji, Xinghu; He, Zhike

2018-06-25

Two kinds of sensitive biosensors based on multi-pedal DNA walker along a 3-D DNA functional magnet particles track for the chemiluminescent detection of streptavidin are constructed and compared in this study. In the presence of SA, multi-pedal DNA walker has been constructed by biotin-modified catalyst as a result of the terminal protection for avoiding the digestion by exonuclease I. Then a toehold of CHA-H1 conjugated with magnetic microparticles (MMPs) could interact with a 'leg' of multi-pedal DNA walker to open the hairpin via toehold-mediated strand exchange catalysis. A newly exposed DNA segment in CHA-H1 would be hybridized with a toehold of biotin-labeled H2. Via the strand displacement process, H2 displaces one 'leg' of multi-pedal DNA walker, and the other 'leg' could still hybridize with neighboring H1 to initiate the next cycle. In order to solve the high background caused by the hybridization between CHA-H1 and H2 without CHA-catalyst, the other model has been designed. The principle of the other model (ISDPR DNA walker) is similar to the above one. After the terminal protection of SA, a 'leg' of multi-pedal DNA walker triggers the opening of the hairpin of ISDPR-H1 conjugated with MMPs. Then the biotin-modified primer could hybridize with the open stem, triggering the polymerization reaction in the presence of dNTPs/polymerase. As the extension of the primer, the 'leg' of multi-pedal DNA walker is displaced so that the other 'leg' could trigger proximal H1 to go on the next cycle. Due to its lower background and stronger signal, multi-pedal DNA walker based on ISDPR has a lower limit of detection for SA. The limit of detection (LOD) for SA is 6.5 pM. What's more, these DNA walker methods have been applied in complex samples successfully.

DNA-Templated Introduction of an Aldehyde Handle in Proteins.

PubMed

Kodal, Anne Louise B; Rosen, Christian B; Mortensen, Michael R; Tørring, Thomas; Gothelf, Kurt V

2016-07-15

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site-specific conjugates. This can rarely be achieved by simple residue-specific random labeling, but generally requires genetic engineering. Using site-selective DNA-templated reductive amination, we created DNA-protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal-binding functionality facilitates site-selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal-binding site. We demonstrate DNA-templated reductive amination for His6 -tagged proteins and metal-binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Replication During Conjugal Transfer of R1162

DTIC Science & Technology

2002-01-01

transport out of the cell. This idea is stimulated by the similarity between the intermediates of single-stranded phage DNA replication and conjugative...the tra gene clusters ( Miele et al., 1991). The protein is not required for replication of the plasmid, and it seems to be dispensable for transfer...donor, in order to reattach to the transport mechanism prior to the start of a new round of transfer. Rolling circle replication itself might also be

CdS nanowires formed by chemical synthesis using conjugated single-stranded DNA molecules

NASA Astrophysics Data System (ADS)

Sarangi, S. N.; Sahu, S. N.; Nozaki, S.

2018-03-01

CdS nanowires were successfully grown by chemical synthesis using two conjugated single-stranded (ss) DNA molecules, poly G (30) and poly C (30), as templates. During the early stage of the synthesis with the DNA molecules, the Cd 2+ interacts with Poly G and Poly C and produces the (Cd 2+)-Poly GC complex. As the growth proceeds, it results in nanowires. The structural analysis by grazing angle x-ray diffraction and transmission electron microscopy confirmed the zinc-blende CdS nanowires with the growth direction of <220>. Although the nanowires are well surface-passivated with the DNA molecules, the photoluminescence quenching was caused by the electron transfer from the nanowires to the DNA molecules. The quenching can be used to detect and label the DNAs.

Conjugates of classical DNA/RNA binder with nucleobase: chemical, biochemical and biomedical applications.

PubMed

Saftic, Dijana; Ban, Zeljka; Matic, Josipa; Tumir, Lidija-Marija; Piantanida, Ivo

2018-05-07

Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class are nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder - nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Tales of conjugation and sex pheromones

PubMed Central

2011-01-01

This review covers highlights of the author's experience becoming and working as a plasmid biologist. The account chronicles a progression from studies of ColE1 DNA in Escherichia coli to Gram-positive bacteria with an emphasis on conjugation in enterococci. It deals with gene amplification, conjugative transposons and sex pheromones in the context of bacterial antibiotic resistance. PMID:22016844

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

PubMed

Kingston, Anthony W; Roussel-Rossin, Chloé; Dupont, Claire; Raleigh, Elisabeth A

2015-01-01

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F'-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10(-12) CFU/recipient per hour.

Nano-Encrypted Morse Code: A Versatile Approach to Programmable and Reversible Nanoscale Assembly and Disassembly

PubMed Central

Wong, Ngo Yin; Xing, Hang; Tan, Li Huey; Lu, Yi

2013-01-01

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code “NANO” and reversible exchange of uppercase letter “I” with lowercase “i”. The yields of the conjugations are high (> 90%) and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates. PMID:23373425

A cationic conjugated polymer and graphene oxide: Application to amplified fluorescence detection of sinapine.

PubMed

Zhang, Zhen; Xiang, Xia; Shi, Jianbin; Huang, Fenghong; Xia, Xiaoyang; Zheng, Mingming; Han, Ling; Tang, Hu

2018-10-05

An amplified fluorescence strategy is described for the detection of sinapine (SP) by using a cationic conjugated polymer (PFP) and graphene oxide (GO). It is observed that the fluorescein (FAM)-labeled single-stranded DNA (FAM-DNA) is absorbed on the surface of GO if SP is absent. This causes that fluorescence resonance energy transfer (FRET) from PFP to FAM is inefficient when adding PFP into FAM-DNA/GO complex. If SP is added to FAM-DNA/GO complex, FAM-DNA is desorbed from GO surface due to the competitive binding of SP and FAM-DNA toward GO. In this case, FAM-DNA is close to PFP in the presence of PFP through strong electrostatic interaction, leading to the occurrence of efficient FRET. Based on the above phenomenon, we demonstrate a method to amplify fluorescence signal of traditional GO-based SP assay by introducing PFP. In comparison to the use of single GO, the combination of PFP with GO-based strategy displays high turn-on ratio and enhanced sensitivity with a limit of detection as low as 7.3 ng mL -1 for SP detection. Satisfactory results in practical samples are also obtained by the recovery experiments, demonstrating the potential application of cationic conjugated polymer in plant-derived small molecule. Copyright © 2018 Elsevier B.V. All rights reserved.

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization.

PubMed

Kavanagh, Paul; Leech, Dónal

2006-04-15

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.

Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

PubMed

Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

2016-01-01

Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences. Copyright © 2015 Elsevier B.V. All rights reserved.

Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

NASA Astrophysics Data System (ADS)

Zhang, Lei; Lei, Dongsheng; Smith, Jessica M.; Zhang, Meng; Tong, Huimin; Zhang, Xing; Lu, Zhuoyang; Liu, Jiankang; Alivisatos, A. Paul; Ren, Gang

2016-03-01

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

PubMed Central

Nishina, Tomoko; Numata, Junna; Nishina, Kazutaka; Yoshida-Tanaka, Kie; Nitta, Keiko; Piao, Wenying; Iwata, Rintaro; Ito, Shingo; Kuwahara, Hiroya; Wada, Takeshi; Mizusawa, Hidehiro; Yokota, Takanori

2015-01-01

We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences. PMID:25584900

Selective alkylation of T–T mismatched DNA using vinyldiaminotriazine–acridine conjugate

PubMed Central

Onizuka, Kazumitsu; Usami, Akira; Yamaoki, Yudai; Kobayashi, Tomohito; Hazemi, Madoka E; Chikuni, Tomoko; Sato, Norihiro; Sasaki, Kaname; Katahira, Masato

2018-01-01

Abstract The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T–T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)–acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T–T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T–T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT–acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1. PMID:29309639

Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor.

PubMed

Boonthum, Chatwalee; Namdee, Katawut; Boonrungsiman, Suwimon; Chatdarong, Kaywalee; Saengkrit, Nattika; Sajomsang, Warayuth; Ponglowhapan, Suppawiwat; Yata, Teerapong

2017-02-10

The main purpose of this study was to investigate the application of modified chitosan as a potential vector for gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells. Such design of gene carrier could be useful in particular for gene therapy for cancers related to the reproductive system, gene disorders of sexual development, and contraception and fertility control. In this study, a decapeptide GnRH was successfully conjugated to chitosan (CS) as confirmed by proton nuclear magnetic resonance spectroscopy ( 1 H NMR) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized GnRH-conjugated chitosan (GnRH-CS) was able to condense DNA to form positively charged nanoparticles and specifically deliver plasmid DNA to targeted cells in both two-dimensional (2D) and three-dimensional (3D) cell cultures systems. Importantly, GnRH-CS exhibited higher transfection activity compared to unmodified CS. In conclusion, GnRH-conjugated chitosan can be a promising carrier for targeted DNA delivery to GnRHR-expressing cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation of arginine modified PEI-conjugated chitosan copolymer for DNA delivery.

PubMed

Zhang, Xi; Duan, Yajing; Wang, Dongfang; Bian, Fengling

2015-05-20

Polyethylenimine-conjugated chitosan (CS-PEI) and arginine modified polyethylenimine-conjugated chitosan (CS-PEI-Arg) were prepared, and the copolymers were characterized by FTIR, (1)H NMR, and XRD. The properties of these copolymers like plasmid DNA (pDNA) binding capacity, complexes' size and zeta potential, cytotoxicity and transfection efficiency were also evaluated. The results show that CS-PEI-Arg derivatives can bind pDNA thoroughly, and form complexes with sizes about 170 nm. Cytotoxicity assay in HepG2 and 293T cells show that CS-PEI-Arg has lower cytotoxicity compared with CS-PEI, which is similar to CS and far below that of PEI. In vitro luciferase assay show that CS-PEI-Arg has better transfection efficiency than CS-PEI, which is superior to that of PEI. The best transfection efficiency of CS-PEI-Arg (N/P = 50) is 2.3-fold, 4.2-fold of those of CS-PEI (N/P = 20) and PEI's (N/P = 10) efficiency respectively. These results display that CS-PEI-Arg is a promising candidate as an efficient gene vector. Copyright © 2014 Elsevier Ltd. All rights reserved.

Noncanonical substrate preference of lambda exonuclease for 5'-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction.

PubMed

Wu, Tongbo; Yang, Yufei; Chen, Wei; Wang, Jiayu; Yang, Ziyu; Wang, Shenlin; Xiao, Xianjin; Li, Mengyuan; Zhao, Meiping

2018-04-06

Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5' non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5' side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π-π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids.

A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water

PubMed Central

Gogoi, Khirud; Mane, Meenakshi V.; Kunte, Sunita S.; Kumar, Vaijayanti A.

2007-01-01

The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ∼3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products. PMID:17981837

Targeting MED1 LxxLL Motifs for Tissue-Selective Treatment of Human Breast Cancer

DTIC Science & Technology

2014-09-01

his colleagues have successfully conjugated malachite green aptamer to RNA nanoparticles characterized by a 3WJ pRNA motif. The in vitro experiment...Folate-DNA/RNA sequence FIGURE 19.5 Diagram of RNA nanoparticle harboring malachite green aptamer, survivin siRNA and folate-DNA/RNA sequence for...405Conjugation of RNA Aptamer to RNA Nanoparticles (Figure 19.5; Shu et al. 2011). The sequence for the malachite green aptamer nanoparticle was rationally

An Efficient Intergeneric Conjugation of DNA from Escherichia coli to Mycelia of the Lincomycin-Producer Streptomyces lincolnensis

PubMed Central

Du, Lei; Liu, Rui-Hua; Ying, Li; Zhao, Guang-Rong

2012-01-01

Streptomyces lincolnensis is a producer of lincomycin, which is a lincosamide antibiotic for the treatment of infective diseases caused by Gram-positive bacteria. S. lincolnensis is refractory to introducing plasmid DNA into cells because of resistance of foreign DNAs and poor sporulation. In this study, a simple and efficient method of transferring plasmids into S. lincolnensis through the intergeneric Escherichia coli-mycelia conjugation was established and optimized for the first time. The recipient mycelia of S. lincolnensis were prepared in liquid SM medium containing 10.3% sucrose for three days. The dispersed mycelia were conjugated with competent E. coli donor cells. The exconjugants were regenerated efficiently on solid mannitol soya flour (MS) medium containing 20 mM MgCl2. The average conjugation frequency was observed at 1.1 × 10−4 per input donor cell and validated functionally by transferring two types of vectors containing lincomycin resistance genes lmrA, lmrB and lmrC into S. lincolnensis mycelia. The data of fermentation in shaking flasks showed the lincomycin yield of the exconjugants increased by 52.9% for the multiple copy vector and 38.3% for the integrative one, compared with the parental strain. The efficient and convenient method of intergeneric E. coli-mycelia conjugation in this study provides a promising procedure to introduce plasmid DNA into other refractory streptomycetes. PMID:22606009

High-yielding and photolabile approaches to the covalent attachment of biomolecules to surfaces via hydrazone chemistry.

PubMed

Lee, Ju Hun; Domaille, Dylan W; Noh, Hyunwoo; Oh, Taeseok; Choi, Chulmin; Jin, Sungho; Cha, Jennifer N

2014-07-22

The development of strategies to couple biomolecules covalently to surfaces is necessary for constructing sensing arrays for biological and biomedical applications. One attractive conjugation reaction is hydrazone formation--the reaction of a hydrazine with an aldehyde or ketone--as both hydrazines and aldehydes/ketones are largely bioorthogonal, which makes this particular reaction suitable for conjugating biomolecules to a variety of substrates. We show that the mild reaction conditions afforded by hydrazone conjugation enable the conjugation of DNA and proteins to the substrate surface in significantly higher yields than can be achieved with traditional bioconjugation techniques, such as maleimide chemistry. Next, we designed and synthesized a photocaged aryl ketone that can be conjugated to a surface and photochemically activated to provide a suitable partner for subsequent hydrazone formation between the surface-anchored ketone and DNA- or protein-hydrazines. Finally, we exploit the latent functionality of the photocaged ketone and pattern multiple biomolecules on the same substrate, effectively demonstrating a strategy for designing substrates with well-defined domains of different biomolecules. We expect that this approach can be extended to the production of multiplexed assays by using an appropriate mask with sequential photoexposure and biomolecule conjugation steps.

Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

2004-12-01

Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

PubMed

Singh, Vijay; Wang, Shenliang; Chan, Ke Min; Clark, Spencer A; Kool, Eric T

2013-04-24

Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

PubMed

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

2017-06-15

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination. Copyright © 2017 American Society for Microbiology.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

PubMed Central

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred

2017-01-01

ABSTRACT Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination. PMID:28411218

Modulating nanoparticle superlattice structure using proteins with tunable bond distributions

DOE PAGES

McMillan, Janet R.; Brodin, Jeffrey D.; Millan, Jaime A.; ...

2017-01-25

Here, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using Beta-galactosidase (βgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein-DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When assembled into crystalline superlattices with AuNPs, we find that the distribution of DNA modifications modulates the favored structure: βgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB 2 packing. We probe the role of protein oligonucleotide number and conjugatemore » size on this observation, which revealed the importance of oligonucleotide distribution and number in this observed assembly behavior. These results indicate that proteins with defined DNA-modification patterns are powerful tools to control the nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein-DNA conjugates.« less

Colorimetric biosensing of targeted gene sequence using dual nanoparticle platforms

PubMed Central

Thavanathan, Jeevan; Huang, Nay Ming; Thong, Kwai Lin

2015-01-01

We have developed a colorimetric biosensor using a dual platform of gold nanoparticles and graphene oxide sheets for the detection of Salmonella enterica. The presence of the invA gene in S. enterica causes a change in color of the biosensor from its original pinkish-red to a light purplish solution. This occurs through the aggregation of the primary gold nanoparticles–conjugated DNA probe onto the surface of the secondary graphene oxide–conjugated DNA probe through DNA hybridization with the targeted DNA sequence. Spectrophotometry analysis showed a shift in wavelength from 525 nm to 600 nm with 1 μM of DNA target. Specificity testing revealed that the biosensor was able to detect various serovars of the S. enterica while no color change was observed with the other bacterial species. Sensitivity testing revealed the limit of detection was at 1 nM of DNA target. This proves the effectiveness of the biosensor in the detection of S. enterica through DNA hybridization. PMID:25897217

A smart magnetic resonance imaging contrast agent responsive to adenosine based on a DNA aptamer-conjugated gadolinium complex.

PubMed

Xu, Weichen; Lu, Yi

2011-05-07

We report a general strategy for developing a smart MRI contrast agent for the sensing of small molecules such as adenosine based on a DNA aptamer that is conjugated to a Gd compound and a protein streptavidin. The binding of adenosine to its aptamer results in the dissociation of the Gd compound from the large protein, leading to decreases in the rotational correlation time and thus change of MRI contrast. © The Royal Society of Chemistry 2011

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

Efficient Fluorescence Resonance Energy Transfer between Quantum Dots and Gold Nanoparticles Based on Porous Silicon Photonic Crystal for DNA Detection.

PubMed

Zhang, Hongyan; Lv, Jie; Jia, Zhenhong

2017-05-10

A novel assembled biosensor was prepared for detecting 16S rRNA, a small-size persistent specific for Actinobacteria. The mechanism of the porous silicon (PS) photonic crystal biosensor is based on the fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and gold nanoparticles (AuNPs) through DNA hybridization, where QDs act as an emission donor and AuNPs serve as a fluorescence quencher. Results showed that the photoluminescence (PL) intensity of PS photonic crystal was drastically increased when the QDs-conjugated probe DNA was adhered to the PS layer by surface modification using a standard cross-link chemistry method. The PL intensity of QDs was decreased when the addition of AuNPs-conjugated complementary 16S rRNA was dropped onto QDs-conjugated PS. Based on the analysis of different target DNA concentration, it was found that the decrease of the PL intensity showed a good linear relationship with complementary DNA concentration in a range from 0.25 to 10 μM, and the detection limit was 328.7 nM. Such an optical FRET biosensor functions on PS-based photonic crystal for DNA detection that differs from the traditional FRET, which is used only in liquid. This method will benefit the development of a new optical FRET label-free biosensor on Si substrate and has great potential in biochips based on integrated optical devices.

Development of programmable small DNA-binding molecules with epigenetic activity for induction of core pluripotency genes.

PubMed

Pandian, Ganesh N; Ohtsuki, Akimichi; Bando, Toshikazu; Sato, Shinsuke; Hashiya, Kaori; Sugiyama, Hiroshi

2012-04-15

Epigenetic modifications that govern the gene expression are often overlooked with the design of artificial genetic switches. N-Methylpyrrole-N-methylimidazole (PI) hairpin polyamides are programmable small DNA binding molecules that have been studied in the context of gene regulation. Recently, we synthesized a library of compounds by conjugating PI polyamides with SAHA, a chromatin-modifier. Among these novel compounds, PI polyamide-SAHA conjugate 1 was shown to epigenetically activate pluripotency genes in mouse embryonic fibroblasts. Here, we report the synthesis of the derivatives of conjugate 1 and demonstrate that these epigenetically active molecules could be developed to improve the induction of pluripotency factors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages and Conjugative Elements

PubMed Central

Iyer, Lakshminarayan M.; Burroughs, A. Maxwell; Anand, Swadha; de Souza, Robson F.

2017-01-01

ABSTRACT Intense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular “weaponry” deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance. IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems. PMID:28559295

Targeting Photoinduced DNA Destruction by Ru(II) Tetraazaphenanthrene in Live Cells by Signal Peptide.

PubMed

Burke, Christopher S; Byrne, Aisling; Keyes, Tia E

2018-06-06

Exploiting NF-κB transcription factor peptide conjugation, a Ru(II)-bis-tap complex (tap = 1,4,5,8-tetraazaphenanthrene) was targeted specifically to the nuclei of live HeLa and CHO cells for the first time. DNA binding of the complex  within the nucleus of live cells was evident from gradual extinction of the metal complex luminescence after it had crossed the nuclear envelope, attributed to guanine quenching of the ruthenium emission via photoinduced electron transfer. Resonance Raman imaging confirmed that the complex remained in the nucleus after emission is extinguished. In the dark and under imaging conditions the cells remain viable, but efficient cellular destruction was induced with precise spatiotemporal control by applying higher irradiation intensities to selected cells. Solution studies indicate that the peptide conjugated complex associates strongly with calf thymus DNA ex-cellulo and gel electrophoresis confirmed that the peptide conjugate is capable of singlet oxygen independent photodamage to plasmid DNA. This indicates that the observed efficient cellular destruction likely operates via direct DNA oxidation by photoinduced electron transfer between guanine and the precision targeted Ru(II)-tap probe. The discrete targeting of polyazaaromatic complexes to the cell nucleus and confirmation that they are photocytotoxic after nuclear delivery is an important step toward their application in cellular phototherapy.

Decitabine Nano-conjugate Sensitizing Human Glioblastoma Cells to Temozolomide

PubMed Central

Cui, Yi; Naz, Asia; Thompson, David H.; Irudayaraj, Joseph

2015-01-01

In this study we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) based nano-conjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells. After synthesis, the highly efficient uptake process and intracellular dynamics of this nano-conjugate was monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nano-vector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing a “positive feedback” to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to excellent internalization and endo-lysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than free drug molecules. Hence, the synthesized nano-conjugate and temozolomide could act in synergy to deliver a more potent and long-term anti-proliferation effect against malignant GBM cells. PMID:25751281

Fluorescent carbon nanoparticle-based lateral flow biosensor for ultrasensitive detection of DNA.

PubMed

Takalkar, Sunitha; Baryeh, Kwaku; Liu, Guodong

2017-12-15

We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioprobes Based on Aptamer and Silica Fluorescent Nanoparticles for Bacteria Salmonella typhimurium Detection

NASA Astrophysics Data System (ADS)

Wang, Qiu-Yue; Kang, Yan-Jun

2016-03-01

In this study, we have developed an efficient method based on single-stranded DNA (ssDNA) aptamers along with silica fluorescence nanoparticles for bacteria Salmonella typhimurium detection. Carboxyl-modified Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (RuBPY)-doped silica nanoparticles (COOH-FSiNPs) were prepared using reverse microemulsion method, and the streptavidin was conjugated to the surface of the prepared COOH-FSiNPs. The bacteria S. typhimurium was incubated with a specific ssDNA biotin-labeled aptamer, and then the aptamer-bacteria conjugates were treated with the synthetic streptavidin-conjugated silica fluorescence nanoprobes (SA-FSiNPs). The results under fluorescence microscopy show that SA-FSiNPs can be applied effectively for the labeling of bacteria S. typhimurium with great photostable property. To further verify the specificity of SA-FSiNPs out of multiple bacterial conditions, variant concentrations of bacteria mixtures composed of bacteria S. typhimurium, Escherichia coli, and Bacillus subtilis were treated with SA-FSiNPs.

A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

PubMed

Wu, Tzong-Yuan; Su, Yi-Yu; Shu, Wei-Hsien; Mercado, Augustus T; Wang, Shi-Kwun; Hsu, Ling-Yi; Tsai, Yow-Fu; Chen, Chung-Yung

2016-04-15

A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced photophysics of conjugated polymers

DOEpatents

Chen, Liaohai [Darien, IL

2007-06-12

A particulate fluorescent conjugated polymer surfactant complex and method of making and using same. The particles are between about 15 and about 50 nm and when formed from a lipsome surfactant have a charge density similar to DNA and are strongly absorbed by cancer cells.

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

PubMed Central

Pokrzywinski, Kaytee L.; Biel, Thomas G.; Kryndushkin, Dmitry; Rao, V. Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis. PMID:28030582

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

PubMed

Pokrzywinski, Kaytee L; Biel, Thomas G; Kryndushkin, Dmitry; Rao, V Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

DOE PAGES

Zhang, Lei; Lei, Dongsheng; Smith, Jessica M.; ...

2016-03-30

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtainmore » 14 density maps at ~ 2-nm resolution . Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.« less

The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus.

PubMed

McMullan, Laura K; Flint, Mike; Dyall, Julie; Albariño, César; Olinger, Gene G; Foster, Scott; Sethna, Phiroze; Hensley, Lisa E; Nichol, Stuart T; Lanier, E Randall; Spiropoulou, Christina F

2016-01-01

Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates. Copyright © 2015. Published by Elsevier B.V.

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

PubMed

Mateos, L M; Schäfer, A; Kalinowski, J; Martin, J F; Pühler, A

1996-10-01

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

PubMed Central

Mateos, L M; Schäfer, A; Kalinowski, J; Martin, J F; Pühler, A

1996-01-01

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure. PMID:8824624

DNA adducts of ethylene dibromide: Aspects of formation and mutagenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cmarik, J.L.

1,2-Dibromoethane (ethylene dibromide, EDB), a potential human carcinogen, undergoes bioactivation by the pathway of glutathione (GSH) conjugation, which generates a reactive intermediate capable of alkylating DNA. The major DNA adduct formed is S-[2-(N[sup 7]-guanyl)ethyl]GSH. This dissertation examined the bioactivation of EDB and the formation of DNA adducts. The selectivity of purified rat and human GSH S-transferases for EDB was examined in vitro. An assay was developed to measure the formation of S,S[prime]-ethylene-bis(GSH). The [alpha] class of the GSH S-transferases was responsible for the majority of EDB-GSH conjugation with both the rat and human enzymes. Human tissue samples for a victimmore » of EDB poisoning were analyzed for S-[2-(N[sup 7]-guanyl)ethyl]GSH utilizing electrochemical detection. No adducts were detected in samples of brain, heart, or kidney. The pattern of alkylation of guanines in fragments of plasmid pBR322 DNA by S-(2-chloroethyl)GSH and related compounds was determined. Alkylation varied approximately ten-fold in intensity and was strongest in runs of guanines. Few differences were observed in the alkylation patterns generated by the different compounds tested. The spectrum of mutations caused by S-(2-chloroethyl)GSH was determined using an M13 bacteriophage forward mutation assay. The majority of mutations (70%) were G:C to A:T transitions. Participation of the N[sup 7]-guanyl adduct in the mutagenic process is strongly implicated. The sequence selectivity of alkylation in the region of M13 sequenced in the mutation assay was determined. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. Sequence context appears to exert a strong influence on the processing of lesions. These studies strongly implicate S-[2-(N[sup 7]-guanyl)-ethyl]GSH as a mutagenic lesion formed by EDB.« less

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

PubMed Central

Yang, Yufei; Chen, Wei; Wang, Jiayu; Yang, Ziyu; Wang, Shenlin; Xiao, Xianjin; Li, Mengyuan

2018-01-01

Abstract Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5′ non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5′ side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π–π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids. PMID:29490081

Toward Single Electron Nanoelectronics Using Self-Assembled DNA Structure.

PubMed

Tapio, Kosti; Leppiniemi, Jenni; Shen, Boxuan; Hytönen, Vesa P; Fritzsche, Wolfgang; Toppari, J Jussi

2016-11-09

DNA based structures offer an adaptable and robust way to develop customized nanostructures for various purposes in bionanotechnology. One main aim in this field is to develop a DNA nanobreadboard for a controllable attachment of nanoparticles or biomolecules to form specific nanoelectronic devices. Here we conjugate three gold nanoparticles on a defined size TX-tile assembly into a linear pattern to form nanometer scale isolated islands that could be utilized in a room temperature single electron transistor. To demonstrate this, conjugated structures were trapped using dielectrophoresis for current-voltage characterization. After trapping only high resistance behavior was observed. However, after extending the islands by chemical growth of gold, several structures exhibited Coulomb blockade behavior from 4.2 K up to room temperature, which gives a good indication that self-assembled DNA structures could be used for nanoelectronic patterning and single electron devices.

Efficient Fluorescence Resonance Energy Transfer between Quantum Dots and Gold Nanoparticles Based on Porous Silicon Photonic Crystal for DNA Detection

PubMed Central

Zhang, Hongyan; Lv, Jie; Jia, Zhenhong

2017-01-01

A novel assembled biosensor was prepared for detecting 16S rRNA, a small-size persistent specific for Actinobacteria. The mechanism of the porous silicon (PS) photonic crystal biosensor is based on the fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and gold nanoparticles (AuNPs) through DNA hybridization, where QDs act as an emission donor and AuNPs serve as a fluorescence quencher. Results showed that the photoluminescence (PL) intensity of PS photonic crystal was drastically increased when the QDs-conjugated probe DNA was adhered to the PS layer by surface modification using a standard cross-link chemistry method. The PL intensity of QDs was decreased when the addition of AuNPs-conjugated complementary 16S rRNA was dropped onto QDs-conjugated PS. Based on the analysis of different target DNA concentration, it was found that the decrease of the PL intensity showed a good linear relationship with complementary DNA concentration in a range from 0.25 to 10 μM, and the detection limit was 328.7 nM. Such an optical FRET biosensor functions on PS-based photonic crystal for DNA detection that differs from the traditional FRET, which is used only in liquid. This method will benefit the development of a new optical FRET label-free biosensor on Si substrate and has great potential in biochips based on integrated optical devices. PMID:28489033

A Comparison of Five Bioconjugatable Ferrocenes for Labeling of Biomolecules†

PubMed Central

Ge, Dongbiao; Levicky, Rastislav

2010-01-01

Five electroactive ferrocene tags for labelling of biomolecules are contrasted with regard to conjugation reactivity with amine and thiol moieties, stability to loss of electrochemical activity, and impact of molecular structure on the redox potential of the free and DNA-conjugated forms. PMID:20740226

Inhibition of the Nedd8 system sensitizes cells to DNA Inter-strand crosslinking agents

PubMed Central

Kee, Younghoon; Huang, Min; Chang, Sophia; Moreau, Lisa A.; Park, Eunmi; Smith, Peter G.; D’Andrea, Alan D.

2012-01-01

The Fanconi Anemia (FA) pathway is required for repair of DNA interstrand crosslinks (ICLs). FA pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as Cisplatin. Conversely, hyperactivation of the FA pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the FA pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL-inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8 conjugating enzymes or treatment with a Nedd8 activating enzyme inhibitor MLN4924, suppressed DNA damage-induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the FA pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells. PMID:22219386

Reinforced self-assembly of donor-acceptor π-conjugated molecules to DNA templates by dipole-dipole interactions together with complementary hydrogen bonding interactions for biomimetics.

PubMed

Yang, Wanggui; Chen, Yali; Wong, Man Shing; Lo, Pik Kwan

2012-10-08

One of the most important criteria for the successful DNA-templated polymerization to generate fully synthetic biomimetic polymers is to design the complementary structural monomers, which assemble to the templates strongly and precisely before carrying polymerization. In this study, water-soluble, laterally thymine-substituted donor-acceptor π-conjugated molecules were designed and synthesized to self-assemble with complementary oligoadenines templates, dA(20) and dA(40), into stable and tubular assemblies through noncovalent interactions including π-π stacking, dipole-dipole interactions, and the complementary adenine-thymine (A-T) hydrogen-bonding. UV-vis, fluorescence, circular dichroism (CD), atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques were used to investigate the formation of highly robust nanofibrous structures. Our results have demonstrated for the first time that the dipole-dipole interactions are stronger and useful to reinforce the assembly of donor-acceptor π-conjugated molecules to DNA templates and the formation of the stable and robust supramolecular nanofibrous complexes together with the complementary hydrogen bonding interactions. This provides an initial step toward DNA-templated polymerization to create fully synthetic DNA-mimetic polymers for biotechnological applications. This study also presents an opportunity to precisely position donor-acceptor type molecules in a controlled manner and tailor-make advanced materials for various biotechnological applications.

Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

NASA Astrophysics Data System (ADS)

Wang, Feng; Liu, Juewen

2014-05-01

Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials. Electronic supplementary information (ESI) available: Methods, additional gels, kinetics, mass spectrum. See DOI: 10.1039/c4nr00867g

Protein/oligonucleotide conjugates as a cell specific PNA carrier.

PubMed

Obara, K; Ishihara, T; Akaike, T; Maruyama, A

2001-01-01

We have focused on proteineus ligand conjugate with oligonucleotides (ODNs) as a cell-specific delivery vector for peptide nucleic acids (PNAs). Asialofetuin (AF), a hepatocyte-specific proteineus ligand, was conjugated with ODNs that served as binding sites for PNAs. Succinimidyl-transe-4(N-maleimidylmethyl)-cyclohexane-1-carboxylate (SMCC) modified AF was coupled with 5'-thiolated oligodeoxynucleotide (HS-ODN). The resulting conjugate held PNAs with sequence-specific manner. The PNA/DNA conjugate complex has resistance against nucleases in serum. The efficient release of PNA from the complex was observed when the complex was made in contact with a target nucleotide. PNA uptake to hepatocytes was greatly enhanced when hepatocytes was incubated with PNA/conjugate complex. Free AF thoroughly inhibited PNA uptake with the conjugate, evidencing asialoglycoprotein receptor (ASGP-R) mediated endocytosis to be a major-route for the cellular uptake.

Toehold-mediated DNA displacement-based surface-enhanced Raman scattering DNA sensor utilizing an Au-Ag bimetallic nanodendrite substrate.

PubMed

Kim, Saetbyeol; Tran Ngoc, Huan; Kim, Joohoon; Yoo, So Young; Chung, Hoeil

2015-07-23

A simple and sensitive surface enhanced Raman scattering (SERS)-based DNA sensor that utilizes the toehold-mediated DNA displacement reaction as a target-capturing scheme has been demonstrated. For a SERS substrate, Au-Ag bimetallic nanodendrites were electrochemically synthesized and used as a sensor platform. The incorporation of both Ag and Au was employed to simultaneously secure high sensitivity and stability of the substrate. An optimal composition of Ag and Au that satisfied these needs was determined. A double-strand composed of 'a probe DNA (pDNA)' complementary to 'a target DNA (tDNA)' and 'an indicator DNA tagged with a Raman reporter (iDNA)' was conjugated on the substrate. The conjugation made the reporter molecule close to the surface and induced generation of the Raman signal. The tDNA released the pre-hybridized iDNA from the pDNA via toehold-mediated displacement, and the displacement of the iDNA resulted in the decrease of Raman intensity. The variation of percent intensity change was sensitive and linear in the concentration range from 200fM to 20nM, and the achieved limit of detection (LOD) was 96.3fM, superior to those reported in previous studies that adopted different signal taggings based on such as fluorescence and electrochemistry. Copyright © 2015 Elsevier B.V. All rights reserved.

METHODS FOR STUDYING BACTERIAL GENE TRANSFER IN SOIL BY CONJUGATION AND TRANSDUCTION

EPA Science Inventory

The purpose of this document is to provide a series of protocols by which a trained technician can conduct studies on the transfer of genetic information by conjugation or transduction in soil, with emphasis on bacteria containing recombinant DNA. The level of the document is gea...

Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Gyoyeon; Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon; Lee, Hansol

The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Ptmore » conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.« less

DNA Conjugation and DNA Directed Self-Assembly of Quantum Dots for Nanophotonic Applications

NASA Astrophysics Data System (ADS)

Samanta, Anirban

Colloidal quantum dots (QDs) or semiconductor nanocrystals are often used to describe 2--20 nm solution processed nanoparticles of various semiconductor materials that display quantum confinement effects. Compared to traditional fluorescent organic dyes, QDs provide many advantages. For biological applications it is necessary to develop reliable methods to functionalize QDs with hydrophilic biomolecules so that they may maintain their stability and functionality in physiological conditions. DNA, a molecule that encodes genetic information, is arguably the smartest molecule that nature has ever produced and one of the most explored bio-macromolecules. QDs that are functionalized with DNA can potentially be organized with nanometer precision by DNA directed self-assembly, and the resulting arrangements may facilitate the display of novel optical properties. The goal of this dissertation was to achieve a robust reliable yet simple strategy to link DNA to QDs so that they can be used for DNA directed self assembly by which we can engineer their optical properties. Presented here is a series of studies to achieve this goal. First we demonstrate the aqueous synthesis of colloidal nanocrystal heterostructures consisting of the CdTe core encapsulated by CdS/ZnS or CdSe/ZnS shells using glutathione (GSH), a tripeptide, as the capping ligand. We next employed this shell synthesis strategy to conjugate PS-PO chimeric DNA to QDs at the time of shell synthesis. We synthesized a library of DNA linked QDs emitting from UV to near IR that are very stable in high salt concentrations. These DNA functionalized QDs were further site-specifically organized on DNA origami in desired patterns directed by DNA self-assembly. We further extended our capability to functionalize DNA to real IR emitting CdxPb 1-xTe alloyed QDs, and demonstrated their stability by self-assembling them on DNA origami. The photo-physical properties of the QDs were further engineered by attaching a QD and a gold nanoparticle in controlled distances on the same DNA origami, which revealed a much longer range quenching effect than usual Forster Resonance Energy Transfer. We are currently engaged in enhancing the photoluminescence intensity of the QDs by bringing them in the plasmonic hot spots generated by a cluster of larger plasmonic nanoparticles.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

PubMed

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

DNA-length-dependent quenching of fluorescently labeled iron oxide nanoparticles with gold, graphene oxide and MoS2 nanostructures.

PubMed

Balcioglu, Mustafa; Rana, Muhit; Robertson, Neil; Yigit, Mehmet V

2014-08-13

We controlled the fluorescence emission of a fluorescently labeled iron oxide nanoparticle using three different nanomaterials with ultraefficient quenching capabilities. The control over the fluorescence emission was investigated via spacing introduced by the surface-functionalized single-stranded DNA molecules. DNA molecules were conjugated on different templates, either on the surface of the fluorescently labeled iron oxide nanoparticles or gold and nanographene oxide. The efficiency of the quenching was determined and compared with various fluorescently labeled iron oxide nanoparticle and nanoquencher combinations using DNA molecules with three different lengths. We have found that the template for DNA conjugation plays significant role on quenching the fluorescence emission of the fluorescently labeled iron oxide nanoparticles. We have observed that the size of the DNA controls the quenching efficiency when conjugated only on the fluorescently labeled iron oxide nanoparticles by setting a spacer between the surfaces and resulting change in the hydrodynamic size. The quenching efficiency with 12mer, 23mer and 36mer oligonucleotides decreased to 56%, 54% and 53% with gold nanoparticles, 58%, 38% and 32% with nanographene oxide, 46%, 38% and 35% with MoS2, respectively. On the other hand, the presence, not the size, of the DNA molecules on the other surfaces quenched the fluorescence significantly with different degrees. To understand the effect of the mobility of the DNA molecules on the nanoparticle surface, DNA molecules were attached to the surface with two different approaches. Covalently immobilized oligonucleotides decreased the quenching efficiency of nanographene oxide and gold nanoparticles to ∼22% and ∼21%, respectively, whereas noncovalently adsorbed oligonucleotides decreased it to ∼25% and ∼55%, respectively. As a result, we have found that each nanoquencher has a powerful quenching capability against a fluorescent nanoparticle, which can be tuned with surface functionalized DNA molecules.

Development of a free-solution SERS-based assay for point-of-care oral cancer biomarker detection using DNA-conjugated gold nanoparticles

NASA Astrophysics Data System (ADS)

Han, Sungyub; Locke, Andrea K.; Oaks, Luke A.; Cheng, Yi-Shing Lisa; Coté, Gerard L.

2018-02-01

It is estimated that the number of new cases of oral cancers worldwide is 529,000 and more than 300,000 deaths each year. The five-year survival rate remains about 50%, and the low survival rate is believed to be due to delayed detection. The primary detection method is through a comprehensive clinical examination by a dentist followed by a biopsy of suspicious lesions. Systematic review and meta-analysis have revealed that clinical examination alone may not be sufficient to cause the clinician to perform a biopsy or refer for biopsy for early detection of OSCC. Therefore, a non-invasive, point-of-Care (POC) detection with high sensitivity and specificity for early detection would be urgently needed, and using salivary biomarkers would be an ideal technology for it. S100 calcium binding protein P (S100P) mRNA presenting in saliva is a potential biomarker for detection of oral cancer. Further, surface enhanced Raman spectroscopy (SERS) has been shown to be a promising POC diagnostic technique. In this research, a SERS-based assay using oligonucleotide strains was developed for the sensitive and rapid detection of S100P. Gold nanoparticles (AuNPs) as a SERS substrate were used for the conjugation with one of two unique 24 base pair oligonucleotides, referred to as left and right DNA probes. A Raman reporter molecule, malachite green isothiocyanate (MGITC), was bound to left-probe-conjugated AuNPs. UV-vis spectroscopy was employed to monitor the conjugation of DNA probes to AuNPs. The hybridization of S100P target to DNA-conjugated AuNPs in sandwich-assay format was confirmed by Raman spectroscopy and shown to yield and R2 of 0.917 across the range of 0-200 nM and a limit of detection of 3 nM.

Structure of Peptide Sex Pheromone Receptor PrgX and PrgX/Pheromone Complexes and Regulation of Conjugation in Enterococcus faecalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi,K.; Brown, C.; Gu, Z.

2005-01-01

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone bindingmore » destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.« less

A single thiazole orange molecule forms an exciplex in a DNA i-motif.

PubMed

Xu, Baochang; Wu, Xiangyang; Yeow, Edwin K L; Shao, Fangwei

2014-06-18

A fluorescent exciplex of thiazole orange (TO) is formed in a single-dye conjugated DNA i-motif. The exciplex fluorescence exhibits a large Stokes shift, high quantum yield, robust response to pH oscillation and little structural disturbance to the DNA quadruplex, which can be used to monitor the folding of high-order DNA structures.

Stepwise Assembly and Characterization of DNA Linked Two-Color Quantum Dot Clusters.

PubMed

Coopersmith, Kaitlin; Han, Hyunjoo; Maye, Mathew M

2015-07-14

The DNA-mediated self-assembly of multicolor quantum dot (QD) clusters via a stepwise approach is described. The CdSe/ZnS QDs were synthesized and functionalized with an amphiphilic copolymer, followed by ssDNA conjugation. At each functionalization step, the QDs were purified via gradient ultracentrifugation, which was found to remove excess polymer and QD aggregates, allowing for improved conjugation yields and assembly reactivity. The QDs were then assembled and disassembled in a stepwise manner at a ssDNA functionalized magnetic colloid, which provided a convenient way to remove unreacted QDs and ssDNA impurities. After assembly/disassembly, the clusters' optical characteristics were studied by fluorescence spectroscopy and the assembly morphology and stoichiometry was imaged via electron microscopy. The results indicate that a significant amount of QD-to-QD energy transfer occurred in the clusters, which was studied as a function of increasing acceptor-to-donor ratios, resulting in increased QD acceptor emission intensities compared to controls.

Inhibition of DNA binding of Sox2 by the SUMO conjugation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuruzoe, Shu; Ishihara, Ko; Uchimura, Yasuhiro

2006-12-29

Sox2 is a member of the high mobility group (HMG) domain DNA-binding proteins for transcriptional control and chromatin architecture. The HMG domain of Sox2 binds the DNA to facilitate transactivation by the cooperative transcription factors such as Oct3/4. We report that mouse Sox2 is modified by SUMO at lysine 247. Substitution of the target lysine to arginine lost the sumoylation but little affected transcriptional potential or nuclear localization of Sox2. By contrast with the unmodified form, Sox2 fused to SUMO-1 did not augment transcription via the Fgf4 enhancer in the presence of Oct3/4. Further, SUMO-1-conjugated Sox2 at the lysine 247more » or at the carboxyl terminus reduced the binding to the Fgf4 enhancer. These indicate that Sox2 sumoylation negatively regulates its transcriptional role through impairing the DNA binding.« less

Potential use of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate (G2) as a DNA carrier in vitro and in vivo.

PubMed

Anno, Takayuki; Higashi, Taishi; Motoyama, Keiichi; Hirayama, Fumitoshi; Uekama, Kaneto; Arima, Hidetoshi

2012-04-01

In this study, we evaluated the polyamidoamine starburst dendrimer (dendrimer, generation 2: G2) conjugate with 6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-cyclodextrin (GUG-β-CDE (G2)) as a gene transfer carrier. The in vitro gene transfer activity of GUG-β-CDE (G2, degree of substitution (DS) of cyclodextrin (CyD) 1.8) was remarkably higher than that of dendrimer (G2) conjugate with α-CyD (α-CDE (G2, DS 1.2)) and that with β-CyD(β-CDE (G2, DS 1.3)) in A549 and RAW264.7 cells. The particle size, ζ-potential, DNase I-catalyzed degradation, and cellular association of plasmid DNA (pDNA) complex with GUG-β-CDE (G2, DS 1.8) were almost the same as those of the other CDEs. Fluorescent-labeled GUG-β-CDE (G2, DS 1.8) localized in the nucleus 6 h after transfection of its pDNA complex in A549 cells, suggesting that nuclear localization of pDNA complex with GUG-β-CDE (G2, DS 1.8), at least in part, contributes to its high gene transfer activity. GUG-β-CDE (G2, DS 1.8) provided higher gene transfer activity than α-CDE (G2, DS 1.2) and β-CDE (G2, DS 1.3) in kidney with negligible changes in blood chemistry values 12 h after intravenous injection of pDNA complexes with GUG-β-CDE (G2, DS 1.8) in mice. In conclusion, the present findings suggest that GUG-β-CDE (G2, DS 1.8) has the potential for a novel polymeric pDNA carrier in vitro and in vivo.

The Key Involvement of Poly(ADP-Ribosyl)ation in Defense Against Toxic Agents: Molecular Biology Studies

DTIC Science & Technology

2008-02-19

compounds, Polyethylenimine (PEI) conjugated to an MLS has also been proposed as a vehicle to deliver DNA to the mitochondria (Lee, et al, 2007). We...100z 50 -, 0 MI.I Codon Probability (%) D RP041 B KANmsft 5COX2 RPO41 3’ CO2 ’ COX2 COX2 WS+X2 pRP041 m 4-4 Z COX2 3’COX2...DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine . J. Drug Target. 15, 115-122. Luisi, P.L

21 CFR 314.70 - Supplements and other changes to an approved application.

Code of Federal Regulations, 2014 CFR

2014-04-01

... derived from such studies; (vi) For a natural product, a recombinant DNA-derived protein/polypeptide, or a...) Changes solely affecting a natural protein, a recombinant DNA-derived protein/polypeptide or a complex or..., recombinant DNA-derived protein/polypeptide, complex or conjugate of a drug substance with a monoclonal...

Promotion of double-duplex invasion of peptide nucleic acids through conjugation with nuclear localization signal peptide.

PubMed

Aiba, Yuichiro; Honda, Yuta; Komiyama, Makoto

2015-03-02

Pseudo-complementary peptide nucleic acid (pcPNA), as one of the most widely used synthetic DNA analogues, invades double-stranded DNA according to Watson-Crick rules to form invasion complexes. This unique mode of DNA recognition induces structural changes at the invasion site and can be used for a range of applications. In this paper, pcPNA is conjugated with a nuclear localization signal (NLS) peptide, and its invading activity is notably promoted both thermodynamically and kinetically. Thus, the double-duplex invasion complex is formed promptly at low pcPNA concentrations under high salt conditions, where the invasion otherwise never occurs. Furthermore, NLS-modified pcPNA is successfully employed for site-selective DNA scission, and the targeted DNA is selectively cleaved under conditions that are not conducive for DNA cutters using unmodified pcPNAs. This strategy of pcPNA modification is expected to be advantageous and promising for a range of in vitro and in vivo applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoinitiator Nucleotide for Quantifying Nucleic Acid Hybridization

PubMed Central

Johnson, Leah M.; Hansen, Ryan R.; Urban, Milan; Kuchta, Robert D.; Bowman, Christopher N.

2010-01-01

This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′-deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm2. Film heights of 10–20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm2 while below the detection limit of ~10 EITC-nucleotides/μm2, no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges. PMID:20337438

New modulated design, docking and synthesis of carbohydrate-conjugate heterobimetallic CuII-SnIV complex as potential topoisomerase II inhibitor: in vitro DNA binding, cleavage and cytotoxicity against human cancer cell lines.

PubMed

Tabassum, Sartaj; Afzal, Mohd; Arjmand, Farukh

2014-03-03

New carbohydrate-conjugate heterobimetallic complexes [C₂₂H₅₀N₆O₁₃CuSnCl₂] (3) and [C₂₂H₅₈N₆O₁₇NiSnCl₂] (4) were synthesized from their monometallic analogs [C₂₂H₅₂N₆O₁₃Cu] (1) and [C₂₂H₆₀N₆O₁₇Ni] (2) containing N-glycoside ligand (L). In vitro DNA binding studies of L and complexes (1-4) with CT DNA were carried out by employing various biophysical and molecular docking techniques which revealed that heterobimetallic complex 3 strongly binds to DNA in comparison to 4, monometallic complexes (1 and 2) and the free ligand. Complex 3 cleaves pBR322 DNA via hydrolytic pathway (confirmed by T4 DNA ligase assay) and inhibited Topo-II activity in a dose-dependent manner. Furthermore, complex 3 was docked into the ATPase domain of human-Topo-II in order to probe the possible mechanism of inhibition. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The fluorescence resonance energy transfer (FRET) gate: a time-resolved study.

PubMed

Xu, Qing-Hua; Wang, Shu; Korystov, Dmitry; Mikhailovsky, Alexander; Bazan, Guillermo C; Moses, Daniel; Heeger, Alan J

2005-01-18

The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB.

The fluorescence resonance energy transfer (FRET) gate: A time-resolved study

PubMed Central

Xu, Qing-Hua; Wang, Shu; Korystov, Dmitry; Mikhailovsky, Alexander; Bazan, Guillermo C.; Moses, Daniel; Heeger, Alan J.

2005-01-01

The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB. PMID:15642946

A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong

2012-11-15

Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges weremore » obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.« less

Characterization of the paclitaxel loaded chitosan graft Pluronic F127 copolymer micelles conjugate with a DNA aptamer targeting HER-2 overexpressing breast cancer cells

NASA Astrophysics Data System (ADS)

Thach Nguyen, Kim; Nguyen, Thu Ha; Do, Dinh Ho; Huan Le, Quang

2017-03-01

In this work we report the isolation of DNA aptamer that is specifically bound to a HER-2 overexpressing SK-BR-3 human breast cancer cell line, using SELEX strategy. Paclitaxel (PTX) loaded chitosan graft Pluronic F127 copolymer micelles conjugate with a DNA aptamer was synthesized and its structure was confirmed by TEM image. This binary mixed system consisting of DNA aptamer modified Pluronic F127 and chitosan could enhance PTX loading capacity and increase micelle stability. Morphology images confirmed the existence of PTX micelles, with an average size of approximately 86.22 ± 1.45 nm diameters. Drug release profile showed that the PTX conjugate maintained a sustained PTX release. From in vitro cell experiment it was shown that 89%-93%, 50%-58%, 55%-62%, 24%-28% and 2%-7% of the SK-BR-3, NS-VN-67, LH-VN-48, HT-VN-26 and NV-VN-31, respectively, were dead after 6-48 h. These results demonstrated a novel DNA aptamer-micelle assembly for efficient detection and a system for the delivery of PTX targeting specific HER-2 overexpressing. We have also successfully cultivated cancer tissues of explants from Vietnamese patients on a type I collagen substrate. The NS-VN-67, LH-VN-48, HT-VN-26 and NV-VN-31cell lines were used as cellular model sources for the study of chemotherapy drug in cancer.

Target binding improves relaxivity in aptamer-gadolinium conjugates.

PubMed

Bernard, Elyse D; Beking, Michael A; Rajamanickam, Karunanithi; Tsai, Eve C; Derosa, Maria C

2012-12-01

MRI contrast agents (CA) have been heavily used over the past several decades to enhance the diagnostic value of the obtained images. From a design perspective, two avenues to improve the efficacy of contrast agents are readily evident: optimization of magnetic properties of the CA, and optimization of the pharmacokinetics and distribution of the CA in the patient. Contrast agents consisting of DNA aptamer-gadolinium(III) conjugates provide a single system in which these factors can be addressed simultaneously. In this proof-of-concept study, the 15mer thrombin aptamer was conjugated to diethylenetriaminepentaacetic (DTPA) dianhydride to form a monoamide derivative of the linear open-chain chelate present in the commonly used contrast agent Magnevist(®). The stability of the conjugated DNA aptamer-DTPA-Gd(III) chelate in a transmetallation study using Zn(II) was found to be similar to that reported for DTPA-Gd(III). Relaxivity enhancements of 35 ± 4 and 20 ± 1 % were observed in the presence of thrombin compared to a control protein at fields of 9.4 and 1.5 T, respectively. The inclusion of spacers between the aptamer and the DTPA to eliminate possible steric effects was also investigated but not found to improve the relaxation enhancement achieved in comparison to the unaltered aptamer conjugate.

DNA-based approaches to the treatment of allergies.

PubMed

Spiegelberg, Hans L; Raz, Eyal

2002-02-01

Although excellent pharmacological treatments for allergies exist, they do not change the underlying pathogenesis of allergic diseases and do not cure the disease. Only allergen-specific immunotherapy, the injection of small but increasing amounts of allergen, has been shown to change a pre-existing allergic Th2 immune response to a non-allergic Th1 response. However, since injection of allergen is associated with the risk of allergic and sometimes even life-threatening anaphylactic reactions, immunotherapy is no longer used as extensively as in the past. In the search for a novel immunotherapy having a low risk-to-benefit ratio, immunostimulatory CpG motif DNA sequences have recently been shown to provide an excellent tool for designing safer and more efficient forms of allergen immunotherapy. These DNA-based immunotherapeutics include allergen gene vaccines, immunization with allergen-DNA conjugates and immunomodulation with immunostimulatory oligodeoxynucleotides. All three DNA-based immunotherapeutics have been shown to be very effective in animal models of allergic diseases and, at present, allergen-DNA conjugates are being tested for their safety and efficacy in allergic patients. This review describes the preclinical findings and the data of the first clinical trials in allergic patients of DNA-based immunotherapeutics for allergic disorders.

Discovery of a conjugative megaplasmid in Bifidobacterium breve.

PubMed

Bottacini, Francesca; O'Connell Motherway, Mary; Casey, Eoghan; McDonnell, Brian; Mahony, Jennifer; Ventura, Marco; van Sinderen, Douwe

2015-01-01

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

PubMed Central

Bottacini, Francesca; O'Connell Motherway, Mary; Casey, Eoghan; McDonnell, Brian; Mahony, Jennifer; Ventura, Marco

2014-01-01

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains. PMID:25326305

Polyfluorophore Excimers and Exciplexes as FRET Donors in DNA

PubMed Central

Teo, Yin Nah; Kool, Eric T.

2009-01-01

We describe studies aimed at testing whether oligomeric exciplex- and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Förster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3’ end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Förster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single wavelength; thus, ODFs may be candidates as “universal FRET donors”, thus allowing multicolor FRET of multiple species to be carried out with one excitation. PMID:19916519

Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA.

PubMed Central

Ihara, T; Maruo, Y; Takenaka, S; Takagi, M

1996-01-01

Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5'-aminohexyl-terminated synthetic oligonucleotides. Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436-443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2-3 kcal mol-1 (1-2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent (with a 10-30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and RNA; single- and double strands) and the sequences. PMID:8932383

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer

PubMed Central

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I.; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase–maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (ΔORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic ΔORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature. PMID:17158161

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer.

PubMed

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

Genomic study of the Type IVC secretion system in Clostridium difficile: understanding C. difficile evolution via horizontal gene transfer.

PubMed

Zhang, Wen; Cheng, Ying; Du, Pengcheng; Zhang, Yuanyuan; Jia, Hongbing; Li, Xianping; Wang, Jing; Han, Na; Qiang, Yujun; Chen, Chen; Lu, Jinxing

2017-01-01

Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.

Rapid and simple G-quadruplex DNA aptasensor with guanine chemiluminescence detection.

PubMed

Cho, Sandy; Park, Lucienne; Chong, Richard; Kim, Young Teck; Lee, Ji Hoon

2014-02-15

Cost-effective and sensitive aptasensor with guanine chemiluminescence detection capable of simply quantifying thrombin in human serum was developed using thrombin aptamer (TBA), one of the G-quadruplex DNA aptamers, without expensive nanoparticles and complicated procedures. Guanines of G-quadruplex TBA-conjugated carboxyfluorescein (6-FAM) bound with thrombin do not react with 3,4,5-trimethoxylphenylglyoxal (TMPG) in the presence of tetra-n-propylammonium hydroxide (TPA), whereas guanines of free TBA- and TBA-conjugated 6-FAM immobilized on the surface of graphene oxide rapidly react with TMPG to emit light. Thus, guanine chemiluminescence in 5% human serum with thrombin was lower than that without thrombin when TBA-conjugated 6-FAM was added in two samples and incubated for 20 min. In other words, the brightness of guanine chemiluminescence was quenched due to the formation of G-quadruplex TBA-conjugated 6-FAM bound with thrombin in a sample. High-energy intermediate, capable of emitting dim light by itself, formed from the reaction between guanines of TBA and TMPG in the presence of TPA, transfers energy to 6-FAM to emit bright light based on the principle of chemiluminescence energy transfer (CRET). G-quadruplex TBA aptasensor devised using the rapid interaction between TBA-conjugated 6-FAM and thrombin quantified trace levels of thrombin without complicated procedures. The limit of detection (LOD = background + 3 × standard deviation) of G-quadruplex TBA aptasensor with good linear calibration curve, accuracy, precision, and recovery was as low as 12.3 nM in 5% human serum. Using the technology reported in this research, we expect that various types of G-quadruplex DNA aptasensors capable of specifically sensing a target molecule such as ATP, HIV, ochratoxin, potassium ions, and thrombin can be developed. © 2013 Elsevier B.V. All rights reserved.

Ligase Detection Reaction for the Analysis of Point Mutations using Free Solution Conjugate Electrophoresis in a Polymer Microfluidic Device

PubMed Central

Sinville, Rondedrick; Coyne, Jennifer; Meagher, Robert J.; Cheng, Yu-Wei; Barany, Francis; Barron, Annelise; Soper, Steven A.

2010-01-01

We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele-specific ligase detection reaction (LDR) with free-solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide “drag-tags” onto the LDR primers. The additional drag of the charge-neutral drag-tag breaks the linear scaling of the charge-to-friction ratio of DNA and enables size-based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag-tags onto a set of four LDR primers designed to probe the K-ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently-labeled LDR/drag-tagged (LDR-dt) products in a multiplexed, single-tube format with mutant:wild-type ratios as low as 1:100, respectively, and the single-base, high-resolution separation of all four LDR-dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a poly(methylmethacrylate), PMMA, microchip replicated via hot-embossing with only a Tris-based running buffer containing additives to suppress the electroosmotic flow (EOF). Typical analysis times for LDR-dt conjugates were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel-based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40. PMID:19053073

Gel Electrophoresis of Gold-DNA Nanoconjugates

DOE PAGES

Pellegrino, T.; Sperling, R. A.; Alivisatos, A. P.; ...

2007-01-01

Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effectivemore » diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.« less

A magnetic nanobead-based bioassay provides sensitive detection of single- and biplex bacterial DNA using a portable AC susceptometer

PubMed Central

Strömberg, Mattias; Zardán Gómez de la Torre, Teresa; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria

2014-01-01

Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin–biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM–NDA further towards implementation in point-of-care and outpatient settings. PMID:24174315

What controls the hybridization thermodynamics of spherical nucleic acids?

PubMed

Randeria, Pratik S; Jones, Matthew R; Kohlstedt, Kevin L; Banga, Resham J; Olvera de la Cruz, Monica; Schatz, George C; Mirkin, Chad A

2015-03-18

The hybridization of free oligonucleotides to densely packed, oriented arrays of DNA modifying the surfaces of spherical nucleic acid (SNA)-gold nanoparticle conjugates occurs with negative cooperativity; i.e., each binding event destabilizes subsequent binding events. DNA hybridization is thus an ever-changing function of the number of strands already hybridized to the particle. Thermodynamic quantification of this behavior reveals a 3 orders of magnitude decrease in the binding constant for the capture of a free oligonucleotide by an SNA conjugate as the fraction of pre-hybridized strands increases from 0 to ∼30%. Increasing the number of pre-hybridized strands imparts an increasing enthalpic penalty to hybridization that makes binding more difficult, while simultaneously decreasing the entropic penalty to hybridization, which makes binding more favorable. Hybridization of free DNA to an SNA is thus governed by both an electrostatic barrier as the SNA accumulates charge with additional binding events and an effect consistent with allostery, where hybridization at certain sites on an SNA modify the binding affinity at a distal site through conformational changes to the remaining single strands. Leveraging these insights allows for the design of conjugates that hybridize free strands with significantly higher efficiencies, some of which approach 100%.

Orientational dynamics and dye-DNA interactions in a dye-labeled DNA aptamer.

PubMed

Unruh, Jay R; Gokulrangan, Giridharan; Lushington, G H; Johnson, Carey K; Wilson, George S

2005-05-01

We report the picosecond and nanosecond timescale rotational dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to immunoglobulin E. Rotational dynamics in combination with fluorescence lifetime measurements provide information about dye-DNA interactions. Comparison of Texas Red (TR), fluorescein, and tetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for biophysical studies employing such conjugates. Time-resolved anisotropy studies demonstrate that the TR- and TAMRA-aptamer anisotropy decays are dominated by the overall rotation of the aptamer, whereas the fluorescein-aptamer anisotropy decay displays a subnanosecond rotational correlation time much shorter than that expected for the overall rotation of the aptamer. Docking and molecular dynamics simulations suggest that the low mobility of TR is a result of binding in the groove of the DNA helix. Additionally, associated anisotropy analysis of the TAMRA-aptamer reveals both quenched and unquenched states that experience significant coupling to the DNA motion. Therefore, quenching of TAMRA by guanosine must depend on the configuration of the dye bound to the DNA. The strong coupling of TR to the rotational dynamics of the DNA aptamer, together with the absence of quenching of its fluorescence by DNA, makes it a good probe of DNA orientational dynamics. The understanding of the nature of dye-DNA interactions provides the basis for the development of bioconjugates optimized for specific biophysical measurements and is important for the sensitivity of anisotropy-based DNA-protein interaction studies employing such conjugates.

Orientational Dynamics and Dye-DNA Interactions in a Dye-Labeled DNA Aptamer

PubMed Central

Unruh, Jay R.; Gokulrangan, Giridharan; Lushington, G. H.; Johnson, Carey K.; Wilson, George S.

2005-01-01

We report the picosecond and nanosecond timescale rotational dynamics of a dye-labeled DNA oligonucleotide or “aptamer” designed to bind specifically to immunoglobulin E. Rotational dynamics in combination with fluorescence lifetime measurements provide information about dye-DNA interactions. Comparison of Texas Red (TR), fluorescein, and tetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for biophysical studies employing such conjugates. Time-resolved anisotropy studies demonstrate that the TR- and TAMRA-aptamer anisotropy decays are dominated by the overall rotation of the aptamer, whereas the fluorescein-aptamer anisotropy decay displays a subnanosecond rotational correlation time much shorter than that expected for the overall rotation of the aptamer. Docking and molecular dynamics simulations suggest that the low mobility of TR is a result of binding in the groove of the DNA helix. Additionally, associated anisotropy analysis of the TAMRA-aptamer reveals both quenched and unquenched states that experience significant coupling to the DNA motion. Therefore, quenching of TAMRA by guanosine must depend on the configuration of the dye bound to the DNA. The strong coupling of TR to the rotational dynamics of the DNA aptamer, together with the absence of quenching of its fluorescence by DNA, makes it a good probe of DNA orientational dynamics. The understanding of the nature of dye-DNA interactions provides the basis for the development of bioconjugates optimized for specific biophysical measurements and is important for the sensitivity of anisotropy-based DNA-protein interaction studies employing such conjugates. PMID:15731389

Steroidogenic acute regulatory protein (StAR) gene expression construct: Development, nanodelivery and effect on reproduction in air-breathing catfish, Clarias batrachus.

PubMed

Rathor, Pravesh Kumar; Bhat, Irfan Ahmad; Rather, Mohd Ashraf; Gireesh-Babu, Pathakota; Kumar, Kundan; Purayil, Suresh Babu Padinhate; Sharma, Rupam

2017-11-01

Steroidogenic acute regulatory protein (StAR) is responsible for the relocation of cholesterol across mitochondrial membrane in vertebrates and is, therefore, a key factor in regulating the rate and timing of steroidogenesis. In the present study, we developed chitosan nanoparticle (CNP) conjugated StAR gene construct (CNP-pcDNA4-StAR) in a eukaryotic expression vector, pcDNA4/HisMax A. CNPs of 135.4nm diameter, 26.7mV zeta potential and 0.381 polydispersity index were used for conjugation. The loading efficiency (LE) of pcDNA4-StAR construct with CNPs was found to be 86%. After the 24h of intramuscular injection, the CNP-pcDNA4-StAR plasmid could be detected from testis, brain, kidney and muscle tissues of Clarias batrachus. The transcript levels of important reproductive genes viz. cyp11a1, cyp17a1, 3β-hsd, 17β-hsd and cyp19a1 in CNP-pcDNA4-StAR treated group were initially low up to 24h, but significantly increased subsequently up to 120h. In naked pcDNA4-StAR treated group, the mRNA level of 3β-hsd, 17β-hsd and cyp19a1 increased initially up to 24h, while cyp11a1 and cyp17a1 increased up to 48h and then started declining. Similar results were obtained for 11-Ketotestosterone and 17β-estradiol. The results indicate relatively long lasting effects of nano-conjugated construct compared to the construct alone. Furthermore, the histopathology of gonads and liver authenticates its possible role in the gonadal development in fish without any adverse effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors.

PubMed

Bacalocostantis, Irene; Mane, Viraj P; Kang, Michael S; Goodley, Addison S; Muro, Silvia; Kofinas, Peter

2012-05-14

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.

Pharmacokinetics and pharmacodynamics of DSTA4637A: A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus in mice

PubMed Central

Zhou, Chenguang; Lehar, Sophie; Gutierrez, Johnny; Rosenberger, Carrie M.; Ljumanovic, Nina; Dinoso, Jason; Koppada, Neelima; Hong, Kyu; Baruch, Amos; Saad, Ola; Mariathasan, Sanjeev; Kamath, Amrita V.

2016-01-01

ABSTRACT DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK. PMID:27653831

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

PubMed

Brun, Sonia; Abella, Neus; Berciano, Maria T; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel; Agell, Neus

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage

PubMed Central

Brun, Sonia; Abella, Neus; Berciano, Maria T.; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus. PMID:28582471

A biotin-conjugated pyridine-based isatoic anhydride, a selective room temperature RNA-acylating agent for the nucleic acid separation.

PubMed

Ursuegui, S; Yougnia, R; Moutin, S; Burr, A; Fossey, C; Cailly, T; Laayoun, A; Laurent, A; Fabis, F

2015-03-28

Isatoic anhydride derivatives, including a biotin and a disulfide linker were specifically designed for nucleic acid separation. 2'-OH selective RNA acylation, capture of biotinylated RNA adducts by streptavidin-coated magnetic beads and disulfide chemical cleavage led to isolation of highly enriched RNA samples from an initial 9/1 DNA-RNA mixture. Starting from the parent compound N-methylisatoic anhydride A which was used at 65 °C, we improved the extraction process by designing a new generation of isatoic anhydrides that are able to react under smoother conditions. Among them, a pyridine-based isatoic anhydride derivative 15f was found to be reactive at room temperature, leading to enhance the efficiency and selectivity of the extraction process by significantly reducing DNA side extraction. The extracted and purified RNAs can then be detected by RT-PCR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baumert, Delphine; Larsen, George; Coopersmith, Kaitlin

A straightforward approach was developed for the synthesis of Pd, Pd-Fe 2O 3, Au-Fe 2O 3, and Au-Pd-Fe 2O 3 nanothermometers, using a single SL DNA. These NP-DNA conjugates were characterized using techniques including EDX measurements, ζ-potential of NPs before and after DNA functionalization, electron microscopy studies and fluorescence spectroscopy. The fluorescence studies of the NP-DNA demonstrate the interaction between the NP and the fluorophore, which is quenched in the case of Au-Pd-Fe 2O 3 NPs and is perhaps enhanced (when compared to AuNPs) in the case of Pd and Pd-Fe 2O 3 NPs. In order to achieve more accuratemore » and reproducible measurements, designing a system that is able to hold the NP-DNA conjugates at a temperature for a longer period of time to allow them to 12 equilibrate is currently underway. Our studies show that Au-Pd-Fe 2O 3 NPs are the best candidate material to serve as nanothermometers when compared to Pd, Pd-Fe 2O 3, and Au-Fe 2O 3 materials.« less

Effect of Backbone Design on Hybridization Thermodynamics of Oligo-nucleic Acids: A Coarse-Grained Molecular Dynamics Simulation Study

NASA Astrophysics Data System (ADS)

Ghobadi, Ahmadreza F.; Jayaraman, Arthi

DNA hybridization is the basis of various bio-nano technologies, such as DNA origami and assembly of DNA-functionalized nanoparticles. A hybridized double stranded (ds) DNA is formed when complementary nucleobases on hybridizing strands exhibit specific and directional hydrogen bonds through canonical Watson-Crick base-pairing interactions. In recent years, the need for cheaper alternatives and significant synthetic advances have driven design of DNA mimics with new backbone chemistries. However, a fundamental understanding of how these backbone modifications in the oligo-nucleic acids impact the hybridization and melting behavior of the duplex is still lacking. In this talk, we present our recent findings on impact of varying backbone chemistry on hybridization of oligo-nucleic acid duplexes. We use coarse-grained molecular dynamics simulations to isolate the effect of strand flexibility, electrostatic interactions and nucleobase spacing on the melting curves for duplexes with various strand sequences and concentrations. Since conjugation of oligo-nucleic acids with polymers serve as building blocks for thermo-responsive polymer networks and gels, we also present the effect of such conjugation on hybridization thermodynamics and polymer conformation.

Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

NASA Astrophysics Data System (ADS)

Khadsai, Sudarat; Rutnakornpituk, Boonjira; Vilaivan, Tirayut; Nakkuntod, Maliwan; Rutnakornpituk, Metha

2016-09-01

Magnetite nanoparticles (MNPs) were surface modified with anionic poly( N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size ( D h) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV-visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation.

PubMed

Kolek, Jan; Sedlar, Karel; Provaznik, Ivo; Patakova, Petra

2016-01-01

Butanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production. We have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 10(2) cfu/μg DNA after step-by-step optimization and an efficiency of 1.6 × 10(2) cfu/μg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 10(2) cfu/μg DNA. Both Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.

Mechanism of Growth Inhibition of Human Cancer Cells by Conjugated Eicosapentaenoic Acid, an Inhibitor of DNA Polymerase and Topoisomerase

PubMed Central

Yonezawa, Yuko; Yoshida, Hiromi; Mizushina, Yoshiyuki

2007-01-01

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We found that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) inhibited the activities of eukaryotic pols and topos in vitro, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC50 values for mammalian pols and human topos of 11.0 – 31.8 and 0.5 – 2.5 μM, respectively. cEPA inhibited the proliferation of two human leukemia cell lines, NALM-6, which is a p53-wild type, and HL-60, which is a p53-null mutant, and the inhibitory effect was stronger than that of normal EPA. In both cell lines, cEPA arrested in the G1 phase, and increased cyclin E protein levels, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. DNA replication-related proteins, such as RPA70, ATR and phosphorylated-Chk1/2, were increased by cEPA treatment in the cell lines, suggesting that cEPA led to DNA replication fork stress inhibiting the activities of pols and topos, and the ATR-dependent DNA damage response pathway could respond to the inhibitor of DNA replication. The compound induced cell apoptosis through both p53-dependent and p53-independent pathways in cell lines NALM-6 and HL-60, respectively. These results suggested the therapeutic potential of conjugated PUFA, such as cEPA, as a leading anti-cancer compound that inhibited pols and topos activities.

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

PubMed

Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B

2014-09-01

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.

Quantification of DNA using the luminescent oxygen channeling assay.

PubMed

Patel, R; Pollner, R; de Keczer, S; Pease, J; Pirio, M; DeChene, N; Dafforn, A; Rose, S

2000-09-01

Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics. Luminescent oxygen channeling assay (LOCI(TM)) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types. In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.

Cellulose-ethylenediaminetetraacetic acid conjugates protect mammalian cells from bacterial cells.

PubMed

Luo, Jie; Lv, Wei; Deng, Ying; Sun, Yuyu

2013-04-08

Cellulose-ethylenediaminetetraacetic acid (EDTA) conjugates were synthesized by the esterification of cellulose with ethylenediaminetetraacetic dianhydride (EDTAD). The new materials provided potent antimicrobial activities against Staphylococcus aureus (S. aureus, Gram-positive bacteria) and Pseudomonas aeruginosa (P. aeruginosa, Gram-negative bacteria), and inhibited the formation of bacterial biofilms. The biocompatibility of the new cellulose-EDTA conjugates was evaluated with mouse skin fibroblasts for up to 14 days. SEM observation and DNA content analysis suggested that the new materials sustained the viability of fibroblast cells. Moreover, in mouse skin fibroblast-bacteria co-culture systems, the new cellulose-EDTA conjugates prevented bacterial biofilm formation and protected the mammalian cells from the bacterial cells for at least one day.

Molecular Engineering of Surfaces for Sensing and Detection

DTIC Science & Technology

2005-08-01

solution was flowed in both chambers at a concentration of 0.05 mg/mL. Biotinylated single- stranded oligonucleotides ( bDNA ) were immobilized on the layer...correspondence between surface-bound bDNA and conjugate, a theoretical minimum coverage of 1.18 × 1012 molecules/cm2 of bDNA is necessary to...immobilize a monolayer of antibody. Above this bDNA coverage a monolayer of immobilized antibody should be observed. These theoretical values are

High density DNA loading on the M13 bacteriophage provides access to colorimetric and fluorescent protein microarray biosensors.

PubMed

Domaille, Dylan W; Lee, Ju Hun; Cha, Jennifer N

2013-02-28

We report the synthesis, characterization, and protein sensing capabilities of M13 bacteriophage-DNA bioconjugates. DNA oligonucleotides were conjugated to M13 through acyl hydrazone linkages. In one case, DNAzymes retained their catalytic ability when anchored to the virus coat, and in a separate study, the dynamic nature of the hydrazone allowed for liberation of DNA from the phage under mild conditions.

Mining the bitter melon (momordica charantia l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

USDA-ARS?s Scientific Manuscript database

Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological tran...

Inducible Alkylation of DNA by a Quinone Methide-Peptide Nucleic Acid Conjugate†

PubMed Central

Liu, Yang; Rokita, Steven E.

2012-01-01

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alkylation of non-complementary sequences is only possible when a template strand is present to co-localize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self adducts formed between the PNA and its attached QM remained active and reversible over more than eight days in aqueous solution prior to reaction with a chosen target added subsequently. PMID:22243337

5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons.

PubMed

Spencer, Jeremy P E; Whiteman, Matthew; Jenner, Peter; Halliwell, Barry

2002-04-01

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

PubMed Central

Stranges, P. Benjamin; Palla, Mirkó; Kalachikov, Sergey; Nivala, Jeff; Dorwart, Michael; Trans, Andrew; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Tao, Chuanjuan; Morozova, Irina; Li, Zengmin; Shi, Shundi; Aberra, Aman; Arnold, Cleoma; Yang, Alexander; Aguirre, Anne; Harada, Eric T.; Korenblum, Daniel; Pollard, James; Bhat, Ashwini; Gremyachinskiy, Dmitriy; Bibillo, Arek; Chen, Roger; Davis, Randy; Russo, James J.; Fuller, Carl W.; Roever, Stefan; Ju, Jingyue; Church, George M.

2016-01-01

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform. PMID:27729524

Self-assembled nanocomplexes of anionic pullulan and polyallylamine for DNA and pH-sensitive intracellular drug delivery

NASA Astrophysics Data System (ADS)

Vora, Lalit; Tyagi, Monica; Patel, Ketan; Gupta, Sanjay; Vavia, Pradeep

2014-12-01

The amalgamation of chemotherapy and gene therapy is promising treatment option for cancer. In this study, novel biocompatible self-assembled nanocomplexes (NCs) between carboxylmethylated pullulan t335 (CMP) with polyallylamine (CMP-PAA NCs) were developed for plasmid DNA (pDNA) and pH-sensitive doxorubicin (DOX) delivery. DOX was conjugated to CMP (DOX-CMP) via hydrazone and confirmed by FTIR and 1H-NMR. In vitro release studies of pH-sensitive DOX-CMP conjugate showed 23 and 85 % release after 48 h at pH 7.4 (physiological pH) and pH 5 (intracellular/tumoral pH), respectively. The CMP-PAA NCs or DOX-CMP-PAA NCs self-assembled into a nanosized (<250 nm) spherical shape as confirmed by DLS and TEM. The hemolysis and cytotoxicity study indicated that the CMP-PAA NCs did not show cytotoxicity in comparison with plain polyallylamine. Gel retardation assay showed complete binding of pDNA with CMP-PAA NCs at 1:2 weight ratio. CMP-PAA NCs/pDNA showed significantly higher transfection in HEK293 cells compared to PAA/pDNA complexes. Confocal imaging demonstrated successful cellular uptake of DOX-CMP-PAA NCs in HEK293 cells. Thus, NCs hold great potential for targeted pDNA and pH-sensitive intratumoral drug delivery.

Synthesis and application of a triazene-ferrocene modifier for immobilization and characterization of oligonucleotides at electrodes.

PubMed

Hansen, Majken N; Farjami, Elaheh; Kristiansen, Martin; Clima, Lilia; Pedersen, Steen Uttrup; Daasbjerg, Kim; Ferapontova, Elena E; Gothelf, Kurt V

2010-04-16

A new DNA modifier containing triazene, ferrocene, and activated ester functionalities was synthesized and applied for electrochemical grafting and characterization of DNA at glassy carbon (GC) and gold electrodes. The modifier was synthesized from ferrocenecarboxylic acid by attaching a phenyltriazene derivative to one of the ferrocene Cp rings, while the other Cp ring containing the carboxylic acid was converted to an activated ester. The modifier was conjugated to an amine-modified DNA sequence. For immobilization of the conjugate at Au or GC electrodes, the triazene was activated by dimethyl sulfate for release of the diazonium salt. The salt was reductively converted to the aryl radical which was readily immobilized at the surface. DNA grafted onto electrodes exhibited remarkable hybridization properties, as detected through a reversible shift in the redox potential of the Fc redox label upon repeated hybridization/denaturation procedures with a complementary target DNA sequence. By using a methylene blue (MB) labeled target DNA sequence the hybridization could also be followed through the MB redox potential. Electrochemical studies demonstrated that grafting through the triazene modifier can successfully compete with existing protocols for DNA immobilization through the commonly used alkanethiol linkers and diazonium salts. Furthermore, the triazene modifier provides a practical one-step immobilization procedure.

Internal-Modified Dithiol DNA-Directed Au Nanoassemblies: Geometrically Controlled Self-Assembly and Quantitative Surface-Enhanced Raman Scattering Properties

NASA Astrophysics Data System (ADS)

Yan, Yuan; Shan, Hangyong; Li, Min; Chen, Shu; Liu, Jianyu; Cheng, Yanfang; Ye, Cui; Yang, Zhilin; Lai, Xuandi; Hu, Jianqiang

2015-11-01

In this work, a hierarchical DNA-directed self-assembly strategy to construct structure-controlled Au nanoassemblies (NAs) has been demonstrated by conjugating Au nanoparticles (NPs) with internal-modified dithiol single-strand DNA (ssDNA) (Au-B-A or A-B-Au-B-A). It is found that the dithiol-ssDNA-modified Au NPs and molecule quantity of thiol-modified ssDNA grafted to Au NPs play critical roles in the assembly of geometrically controlled Au NAs. Through matching Au-DNA self-assembly units, geometrical structures of the Au NAs can be tailored from one-dimensional (1D) to quasi-2D and 2D. Au-B-A conjugates readily give 1D and quasi-2D Au NAs while 2D Au NAs can be formed by A-B-Au-B-A building blocks. Surface-enhanced Raman scattering (SERS) measurements and 3D finite-difference time domain (3D-FDTD) calculation results indicate that the geometrically controllable Au NAs have regular and linearly “hot spots”-number-depended SERS properties. For a certain number of NPs, the number of “hot spots” and accordingly enhancement factor of Au NAs can be quantitatively evaluated, which open a new avenue for quantitative analysis based on SERS technique.

Fueling and Stabilizing a Biomolecular Motor-Powered Biosensor for Remote Detection Scenarios

DTIC Science & Technology

2007-10-01

streptavidin, cyclodextrin host - guest 0 , malachite green - aptamer", DNA - DNA12, and antibody - antigen . Using functionalized microtubules gliding on...3), 646-648 (2005). 11 Hirabayashi, M. et al. Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2017-05-24

A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labeled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy) 3 2+ -conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H 2 O 2 in situ. The ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by the presence of H 2 O 2 . By integrating the programmable DNA cyclic amplification and in situ generating H 2 O 2 as Ru(bpy) 3 2+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labeling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity and good capturing efficiency of tetrahedral-decorated electrode surface but also provides potential applications in the construction of ECL biosensor.

Enzymatic production of single-molecule FISH and RNA capture probes.

PubMed

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

pSK41-Like Plasmid Is Necessary for Inc18-Like vanA Plasmid Transfer from Enterococcus faecalis to Staphylococcus aureus In Vitro

PubMed Central

Clark, Nancye; Patel, Jean B.

2013-01-01

Vancomycin-resistant Staphylococcus aureus (VRSA) is thought to result from the in vivo conjugative transfer of a vanA plasmid from an Enterococcus sp. to S. aureus. We studied bacterial isolates from VRSA cases that occurred in the United States to identify microbiological factors which may contribute to this plasmid transfer. First, vancomycin-susceptible, methicillin-resistant S. aureus (MRSA) isolates from five VRSA cases were tested for their ability to accept foreign DNA by conjugation in mating experiments with Enterococcus faecalis JH2-2 containing pAM378, a pheromone-response conjugative plasmid. All of the MRSA isolates accepted the plasmid DNA with similar transfer efficiencies (∼10−7/donor CFU) except for one isolate, MRSA8, for which conjugation was not successful. The MRSA isolates were also tested as recipients in mating experiments between an E. faecalis isolate with an Inc18-like vanA plasmid that was isolated from a VRSA case patient. Conjugative transfer was successful for 3/5 MRSA isolates. Successful MRSA recipients carried a pSK41-like plasmid, a staphylococcal conjugative plasmid, whereas the two unsuccessful MRSA recipients did not carry pSK41. The transfer of a pSK41-like plasmid from a successful MRSA recipient to the two unsuccessful recipients resulted in conjugal transfer of the Inc18-like vanA plasmid from E. faecalis at a frequency of 10−7/recipient CFU. In addition, conjugal transfer could be achieved for pSK41-negative MRSA in the presence of a cell-free culture filtrate from S. aureus carrying a pSK41-like plasmid at a frequency of 10−8/recipient CFU. These results indicated that a pSK41-like plasmid can facilitate the transfer of an Inc18-like vanA plasmid from E. faecalis to S. aureus, possibly via an extracellular factor produced by pSK41-carrying isolates. PMID:23089754

Guanidinylated polyethyleneimine-polyoxypropylene-polyoxyethylene conjugates as gene transfection agents.

PubMed

Bromberg, Lev; Raduyk, Svetlana; Hatton, T Alan; Concheiro, Angel; Rodriguez-Valencia, Cosme; Silva, Maite; Alvarez-Lorenzo, Carmen

2009-05-20

Conjugates of linear and branched polyethyleneimine (PEI) and monoamine polyether Jeffamine M-2070 (PO/EO mol ratio 10/31, 2000 Da) were synthesized through polyether activation by cyanuric chloride followed by attachment to PEI and guanidinylation by 1H-pyrazole-carboxamidine hydrochloride. The resulting guanidinylated PEI-polyether conjugates (termed gPEI-Jeffamine) efficiently complexed plasmid DNA, and their polyplexes possessed enhanced colloidal stability in the presence of serum proteins. In vitro studies with mammalian CHO-1, 3T3, and Cos-7 cell lines demonstrated improved transfection efficiency of the pCMVbeta-gal plasmid/gPEI-Jeffamine polyplexes. The guanidinylation of the amino groups of PEI and the conjugation of PEI with the Jeffamine polyether enhanced the conjugates' interaction with genetic material and reduced the cytotoxicity of the polyplexes in experiments with the L929 cell line.

Synthesis and characterization of PEG-conjugated quaternized chitosan and its application as a gene vector.

PubMed

Zhang, Xi; Yao, Juan; Zhang, Lihong; Fang, Jianguo; Bian, Fengling

2014-03-15

Poly(ethylene glycol)-conjugated N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride (PHTAC) derivatives were prepared by incorporating PEG molecules onto quaternized chitosan backbone. The copolymers were characterized by FTIR, (1)H NMR and XRD. Agarose gel retardation assay indicated that PHTAC had good plasmid DNA (pDNA) binding capability and the particle sizes of PHTAC/pDNA complexes determined by DLS were about 200 nm. Cytotoxicity assays in HeLa and 293T cells showed that PHTAC had low cytotoxicity. In vitro luciferase assay showed that PHTAC with PEGylation degree of 9% (PHTAC-1) had good transfection efficiency about 5.3-fold higher than quaternized chitosan, which was comparable with PEI (25 kDa). These results suggest that PHTAC-1 is a promising candidate as an efficient nonviral gene vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification and Application of Plasmids Suitable for Transfer of Foreign DNA to Members of the Genus Gordonia

PubMed Central

Arenskötter, Matthias; Baumeister, Dirk; Kalscheuer, Rainer; Steinbüchel, Alexander

2003-01-01

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 × 105 CFU/μg of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499T, G. rubropertincta DSM43197T, G. rubropertincta DSM46038, and G. terrae DSM43249T. Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 × 10−6 of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed. PMID:12902293

Phase-change related epigenetic and physiological changes in Pinus radiata D. Don.

PubMed

Fraga, Mario F; Cañal, Maria Jesús; Rodríguez, Roberto

2002-08-01

DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30-35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.

Light-induced propulsion of a giant liposome driven by peptide nanofibre growth.

PubMed

Inaba, Hiroshi; Uemura, Akihito; Morishita, Kazushi; Kohiki, Taiki; Shigenaga, Akira; Otaka, Akira; Matsuura, Kazunori

2018-04-19

Light-driven nano/micromotors are attracting much attention, not only as molecular devices but also as components of bioinspired robots. In nature, several pathogens such as Listeria use actin polymerisation machinery for their propulsion. Despite the development of various motors, it remains challenging to mimic natural systems to create artificial motors propelled by fibre formation. Herein, we report the propulsion of giant liposomes driven by light-induced peptide nanofibre growth on their surface. Peptide-DNA conjugates connected by a photocleavage unit were asymmetrically introduced onto phase-separated giant liposomes. Ultraviolet (UV) light irradiation cleaved the conjugates and released peptide units, which self-assembled into nanofibres, driving the translational movement of the liposomes. The velocity of the liposomes reflected the rates of the photocleavage reaction and subsequent fibre formation of the peptide-DNA conjugates. These results showed that chemical design of the light-induced peptide nanofibre formation is a useful approach to fabricating bioinspired motors with controllable motility.

Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

PubMed

Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

2015-06-05

To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island

PubMed Central

Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao

2016-01-01

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICEAc) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICEAc-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity. PMID:27849579

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

PubMed

Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao; Zhu, Jun

2016-11-29

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICE Ac ) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICE Ac -located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

PubMed

He, Bingwei; Wang, Yitong; Shao, Naimin; Chang, Hong; Cheng, Yiyun

2015-08-01

Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mannose-pepstatin conjugates as targeted inhibitors of antigen processing.

PubMed

Free, Paul; Hurley, Christopher A; Kageyama, Takashi; Chain, Benjamin M; Tabor, Alethea B

2006-05-07

The molecular details of antigen processing, including the identity of the enzymes involved, their intracellular location and their substrate specificity, are still incompletely understood. Selective inhibition of proteolytic antigen processing enzymes such as cathepsins D and E, using small molecular inhibitors such as pepstatin, has proven to be a valuable tool in investigating these pathways. However, pepstatin is poorly soluble in water and has limited access to the antigen processing compartment in antigen presenting cells. We have synthesised mannose-pepstatin conjugates, and neomannosylated BSA-pepstatin conjugates, as tools for the in vivo study of the antigen processing pathway. Conjugation to mannose and to neomannosylated BSA substantially improved the solubility of the conjugates relative to pepstatin. The mannose-pepstatin conjugates showed no reduction in inhibition of cathepsin E, whereas the neomannosylated BSA-pepstatin conjugates showed some loss of inhibition, probably due to steric factors. However, a neomannosylated BSA-pepstatin conjugate incorporating a cleavable disulfide linkage between the pepstatin and the BSA showed the best uptake to dendritic cells and the best inhibition of antigen processing.

ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans.

PubMed

Bustamante, Paula; Covarrubias, Paulo C; Levicán, Gloria; Katz, Assaf; Tapia, Pablo; Holmes, David; Quatrini, Raquel; Orellana, Omar

2012-01-01

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation. Copyright © 2013 S. Karger AG, Basel.

Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

PubMed

Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

2015-10-01

Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Uncovering the self-assembly of DNA nanostructures by thermodynamics and kinetics.

PubMed

Wei, Xixi; Nangreave, Jeanette; Liu, Yan

2014-06-17

CONSPECTUS: DNA nanotechnology is one of the most flourishing interdisciplinary research fields. DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales. They can be used as scaffolds for organizing other nanoparticles, proteins, and chemical groups, leveraging their functions for creating complex bioinspired materials that may serve as smart drug delivery systems, in vitro or in vivo biomolecular computing platforms, and diagnostic devices. Achieving optimal structural features, efficient assembly protocols, and precise functional group positioning and modification requires a thorough understanding of the thermodynamics and kinetics of the DNA nanostructure self-assembly process. The most common real-time measurement strategies include monitoring changes in UV absorbance based on the hyperchromic effect of DNA, and the emission signal changes of DNA intercalating dyes or covalently conjugated fluorescent dyes/pairs that accompany temperature dependent structural changes. Thermodynamic studies of a variety of DNA nanostructures have been performed, from simple double stranded DNA formation to more complex origami assembly. The key parameters that have been evaluated in terms of stability and cooperativity include the overall dimensions, the folding path of the scaffold, crossover and nick point arrangement, length and sequence of single strands, and salt and ion concentrations. DNA tile-tile interactions through sticky end hybridization have also been analyzed, and the steric inhibition and rigidity of tiles turn out to be important factors. Many kinetic studies have also been reported, and most are based on double stranded DNA formation. A two-state assumption and the hypothesis of several intermediate states have been applied to determine the rate constant and activation energy of the DNA hybridization process. A few simulated models were proposed to represent the structural, mechanical, and kinetic properties of DNA hybridization. The kinetics of strand displacement reactions has also been studied as a special case of DNA hybridization. The thermodynamic and kinetic characteristics of DNA nanostructures have been exploited to develop rapid and isothermal annealing protocols. It is conceivable that a more thorough understanding of the DNA assembly process could be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications.

Discovery of a new family of relaxases in Firmicutes bacteria.

PubMed

Ramachandran, Gayetri; Miguel-Arribas, Andrés; Abia, David; Singh, Praveen K; Crespo, Isidro; Gago-Córdoba, César; Hao, Jian An; Luque-Ortega, Juan Roman; Alfonso, Carlos; Wu, Ling J; Boer, D Roeland; Meijer, Wilfried J J

2017-02-01

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.

Discovery of a new family of relaxases in Firmicutes bacteria

PubMed Central

Singh, Praveen K.; Hao, Jian An; Luque-Ortega, Juan Roman; Wu, Ling J.; Boer, D. Roeland

2017-01-01

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research. PMID:28207825

Preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices

PubMed Central

Powell, Richard D.; Hainfeld, James F.

2013-01-01

Nanogold and undecagold are covalently linked gold cluster labels which enable the identification and localization of biological components with molecular precision and resolution. They can be prepared with different reactivities, which means they can be conjugated to a wide variety of molecules, including nucleic acids, at specific, unique sites. The location of these sites can be synthetically programmed in order to preserve the binding affinity of the conjugate and impart novel characteristics and useful functionality. Methods for the conjugation of undecagold and Nanogold to DNA and RNA are discussed, and applications of labeled conjugates to the high-resolution microscopic identification of binding sites and characterization of biological macromolecular assemblies are described. In addition to providing insights into their molecular structure and function, high-resolution microscopic methods also show how Nanogold and undecagold conjugates can be synthetically assembled, or self-assemble, into supramolecular materials to which the gold cluster labels impart useful functionality. PMID:20869258

In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency.

PubMed Central

Davis, R; Vapnek, D

1976-01-01

The amounts of plasmid deoxyribonucleic acid (DNA) and the levels of the in vivo transcription of the Escherichia coli plasmids R538-1 (repressed for conjugal transfer) and R538-1drd (derepressed for transfer) were determined by DNA-DNA hybridization and DNA-ribonucleic acid hybridization, respectively. The results demonstrate that the level of plasmid transcription is increased by two-fold in the strain carrying the derepressed plasmid, compared to an isogenic strain carrying the repressed plasmid, whereas the amount of plasmid DNA is approximately the same, suggesting that the transfer genes are under transcriptional control. Levels of plasmid DNA, plasmid DNA transcription, and chloramphenicol acetyltransferase activity were also compared in a mutant strain that carried the R538-1drd plasmid and was resistant to high levels of antibiotics. This strain produces about 13 copies of plasmid DNA per chromosome compared to five copies for the parent strain. The level of transcription of plasmid DNA was found to be twofold higher in the high-level resistant strain, whereas the level of chloramphenition, acetyltransferase activity was increased by 10-fold. In addition the levels of plasmid DNA transcription and chloramphenicol acetyltransferase activity in the high-level resistant strain were found to be further increased by the presence of high levels of chloramphenicol in the growth medium. The amount of plasmid DNA remained constant under these conditions, indicating that high levels of chloramphenicol can stimulate the expression of plasmid genes at the level of transcription in this strain. PMID:767321

Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

NASA Astrophysics Data System (ADS)

Tomiyama, Tetsuro; Toita, Riki; Kang, Jeong-Hun; Koga, Haruka; Shiosaki, Shujiro; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

2011-09-01

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

Aptamer-conjugated DNA nano-ring as the carrier of drug molecules

NASA Astrophysics Data System (ADS)

Srivithya, Vellampatti; Roun, Heo; Sekhar Babu, Mitta; Hyung, Park Jae; Ha, Park Sung

2018-03-01

Due to its predictable self-assembly and structural stability, structural DNA nanotechnology is considered one of the main interdisciplinary subjects encompassing conventional nanotechnology and biotechnology. Here we have fabricated the mucin aptamer (MUC1)˗conjugated DNA nano˗ring intercalated with doxorubicin (DNRA˗DOX) as potential therapeutics for breast cancer. DNRA˗DOX exhibited significantly higher cytotoxicity to the MCF˗7 breast cancer cells than the controls, including DOX alone and the aptamer deficient DNA nano˗ring (DNR) with doxorubicin. Interactions between DOX and DNRA were studied using spectrophotometric measurements. Dose-dependent cytotoxicity was performed to prove that both DNR and DNRA were non-toxic to the cells. The drug release profile showed a controlled release of DOX at normal physiological pH 7.4, with approximately 61% released, but when exposed to lysosomal of pH 5.5, the corresponding 95% was released within 48 h. Owing to the presence of the aptamer, DNRA˗DOX was effectively taken up by the cancer cells, as confirmed by confocal microscopy, implying that it has potential for use in targeted drug delivery.

Direct Nanoscale Conversion of Biomolecular Signals into Electronic Information

DTIC Science & Technology

2008-09-22

the electrode surface. In this experiment, the single free cysteine group featured in the GOx structure was exploited to demonstrate that orientation...first with GOx-ssDNA conjugates featuring a sequence complementary to the address strand, then with a non-complementary conjugate and finally with...fully-functional for an enzyme that features a free thiol group, or that can be engineered to incorporate a thiol onto its outer shell

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA.

PubMed

Matthew Whiteman Andrew Jenner Barry Halliwell

1999-01-01

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.

Functionalization of quantum rods with oligonucleotides for programmable assembly with DNA origami

NASA Astrophysics Data System (ADS)

Doane, Tennyson L.; Alam, Rabeka; Maye, Mathew M.

2015-02-01

The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions.The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions. Electronic supplementary information (ESI) available: Experimental conditions, DNA origami blueprint and sequences, FRET calculations. Additional Fig. S1-S13. See DOI: 10.1039/c4nr07662a

Anti-TROP2 conjugated hollow gold nanospheres as a novel nanostructure for targeted photothermal destruction of cervical cancer cells

NASA Astrophysics Data System (ADS)

Liu, Ting; Tian, Jiguang; Chen, Zhaolong; Liang, Ying; Liu, Jiao; Liu, Si; Li, Huihui; Zhan, Jinhua; Yang, Xingsheng

2014-08-01

Photothermal ablation (PTA) is a promising avenue in the area of cancer therapeutics that destroys tumor cells through conversion of near-infrared (NIR) laser light to heat. Hollow gold nanospheres (HGNs) are one of the few materials that are capable of converting light to heat and have been previously used for photothermal ablation studies. Selective delivery of functional nanoparticles to the tumor site is considered as an effective therapeutic approach. In this paper, we demonstrated the anti-cancer potential of HGNs. HGNs were conjugated with monoclonal antibody (anti-TROP2) in order to target cervical cancer cells (HeLa) that contain abundant trophoblast cell surface antigen 2 (TROP2) on the cell surface. The efficient uptake and intracellular location of these functionalized HGNs were studied through application of inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy (TEM). Cytotoxicity induced by PTA was measured using CCK-8 assay. HeLa cells incubated with naked HGNs (0.3-3 nmol L-1) within 48 h did not show obvious cytotoxicity. Under laser irradiation at suitable power, anti-TROP2 conjugated HGNs achieved significant tumor cell growth inhibition in comparison to the effects of non-specific PEGylated HGNs (P < 0.05). γH2AX assay results revealed higher occurrences of DNA-DSBs with anti-TROP2 conjugated HGNs plus laser radiation as compared to treatment with laser alone. Flow cytometry analysis showed that the amount of cell apoptosis was increased after laser irradiation with anti-TROP2 conjugated HGNs (P < 0.05). Anti-TROP2 conjugated HGNs resulted in down-regulation of Bcl-2 expression and up-regulation of Bax expression. Our study results confirmed that anti-TROP2 conjugated HGNs can selectively destroy cervical cancer cells through inducing its apoptosis and DNA damages. We propose that HGNs have the potentials to mediate targeted cancer treatment.

Combining Chemoselective Ligation with Polyhistidine-Driven Self-Assembly for the Modular Display of Biomolecules on Quantum Dots

PubMed Central

Prasuhn, Duane E.; Blanco-Canosa, Juan B.; Vora, Gary J.; Delehanty, James B.; Susumu, Kimihiro; Mei, Bing C.; Dawson, Philip E.; Medintz, Igor L.

2015-01-01

One of the principle hurdles to wider incorporation of semiconductor quantum dots (QDs) in biology is the lack of facile linkage chemistries to create different types of functional QD-bioconjugates. A two-step modular strategy for the presentation of biomolecules on CdSe/ZnS core/shell QDs is described here which utilizes a chemoselective, aniline-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto peptides and DNA. This specifically provides them the ability to ratiometrically self-assemble to hydrophilic QDs. The versatility of this labeling approach was highlighted by ligating proteolytic substrate peptides, an oligoarginine cell-penetrating peptide, or a DNA-probe to cognate hexahistidine peptidyl sequences. The modularity allowed subsequently self-assembled QD constructs to engage in different types of targeted bioassays. The self-assembly and photophysical properties of individual QD conjugates were first confirmed by gel electrophoresis and Förster resonance energy transfer analysis. QD-dye-labeled peptide conjugates were then used as biosensors to quantitatively monitor the proteolytic activity of caspase-3 or elastase enzymes from different species. These sensors allowed the determination of the corresponding kinetic parameters, including the Michaelis constant (KM) and the maximum proteolytic activity (Vmax). QDs decorated with cell-penetrating peptides were shown to be successfully internalized by HEK 293T/17 cells, while nanocrystals displaying peptide-DNA conjugates were utilized as fluorescent probes in hybridization microarray assays. This modular approach for displaying peptides or DNA on QDs may be extended to other more complex biomolecules such as proteins or utilized with different types of nanoparticle materials. PMID:20099912

Synthesis of a multi-functional DNA nanosphere barcode system for direct cell detection.

PubMed

Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

2017-09-28

Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling circle amplification (RCA) with several functionalized nucleotides. DNANSs with a barcode system were designed to exhibit fluorescence for coding enhanced signals and contain biotin for more functionalities, including targeting through the biotin-streptavidin (biotin-STA) interaction. Functionalized deoxynucleotide triphosphates (dNTPs) were mixed in the RCA process and functional moieties can be expressed on the DNANSs. The anti-epidermal growth factor receptor antibodies (anti-EGFR Abs) can be conjugated on DNANSs for targeting cancer cells specifically. As a proof of concept, the potential of the multi-functional DNANS barcode was demonstrated by direct cell detection as a simple detection method. The DNANS barcode provides a new route for the simple and rapid selective recognition of cancer cells.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.

Antimicrobial resistance inStaphylococcus aureuspresents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriTregion of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by themore » full 665-residue NES proteinin vitro. Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureusplasmids that contain sequences similar to theoriTregion of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriTsequences of these nonconjugative plasmidsin vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriTmimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT. These data indicate that the conjugative relaxase intransmechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. IMPORTANCEUnderstanding the mechanism of antimicrobial resistance transfer in bacteria such asStaphylococcus aureusis an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of theStaphylococcus aureusmultiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variantoriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase intransmechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.« less

Specificity and disease in the ubiquitin system

PubMed Central

Chaugule, Viduth K.; Walden, Helen

2016-01-01

Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

Folding DNA into a Lipid-Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins.

PubMed

Dong, Yuanchen; Chen, Shuobing; Zhang, Shijian; Sodroski, Joseph; Yang, Zhongqiang; Liu, Dongsheng; Mao, Youdong

2018-02-19

Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nanobarrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-molecule optical-trapping measurements with DNA anchored to an array of gold nanoposts.

PubMed

Paik, D Hern; Perkins, Thomas T

2012-01-01

Gold-thiol chemistry is one of the most successful chemistries for conjugating biomolecules to surfaces, but such chemistry has not been exploited in optical-trapping experiments because of laser-induced ablation of gold. In this work, we describe a method to combine these two separate technologies without undue heating using DNA anchored to gold nanostructures (r = 50-250 nm; h ≈ 20 nm). Moreover, we demonstrate a quantitative and mechanically robust (>100 pN) optical-trapping assay. By using three dithiol phosphoramidites (DTPAs) incorporated into a polymerase chain reaction (PCR) primer, the gold-DNA bond remained stable in the presence of excess thiolated compounds. This chemical robustness allowed us to reduce nonspecific sticking by passivating the unreacted gold with methoxy-(polyethylene glycol)-thiol (mPEG-SH). Overall, this surface conjugation of biomolecules onto an ordered array of gold nanostructures by chemically and mechanically robust bonds provides a unique way to carry out spatially controlled, repeatable measurements of single molecules.

Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart

NASA Astrophysics Data System (ADS)

Khan, Raffay S.; Martinez, Mario D.; Sy, Jay C.; Pendergrass, Karl D.; Che, Pao-Lin; Brown, Milton E.; Cabigas, E. Bernadette; Dasari, Madhuri; Murthy, Niren; Davis, Michael E.

2014-03-01

There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.

A Synthetic DNA-Binding Domain Guides Distinct Chromatin-Modifying Small Molecules to Activate an Identical Gene Network.

PubMed

Han, Le; Pandian, Ganesh N; Chandran, Anandhakumar; Sato, Shinsuke; Taniguchi, Junichi; Kashiwazaki, Gengo; Sawatani, Yoshito; Hashiya, Kaori; Bando, Toshikazu; Xu, Yufang; Qian, Xuhong; Sugiyama, Hiroshi

2015-07-20

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integrative and conjugative elements and their hosts: composition, distribution and organization

PubMed Central

Touchon, Marie; Rocha, Eduardo P. C.

2017-01-01

Abstract Conjugation of single-stranded DNA drives horizontal gene transfer between bacteria and was widely studied in conjugative plasmids. The organization and function of integrative and conjugative elements (ICE), even if they are more abundant, was only studied in a few model systems. Comparative genomics of ICE has been precluded by the difficulty in finding and delimiting these elements. Here, we present the results of a method that circumvents these problems by requiring only the identification of the conjugation genes and the species’ pan-genome. We delimited 200 ICEs and this allowed the first large-scale characterization of these elements. We quantified the presence in ICEs of a wide set of functions associated with the biology of mobile genetic elements, including some that are typically associated with plasmids, such as partition and replication. Protein sequence similarity networks and phylogenetic analyses revealed that ICEs are structured in functional modules. Integrases and conjugation systems have different evolutionary histories, even if the gene repertoires of ICEs can be grouped in function of conjugation types. Our characterization of the composition and organization of ICEs paves the way for future functional and evolutionary analyses of their cargo genes, composed of a majority of unknown function genes. PMID:28911112

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

PubMed

Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

2018-06-04

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

Label-Free Fluorescence Assay of S1 Nuclease and Hydroxyl Radicals Based on Water-Soluble Conjugated Polymers and WS₂ Nanosheets.

PubMed

Li, Junting; Zhao, Qi; Tang, Yanli

2016-06-13

We developed a new method for detecting S1 nuclease and hydroxyl radicals based on the use of water-soluble conjugated poly[9,9-bis(6,6-(N,N,N-trimethylammonium)-fluorene)-2,7-ylenevinylene-co-alt-2,5-dicyano-1,4-phenylene)] (PFVCN) and tungsten disulfide (WS₂) nanosheets. Cationic PFVCN is used as a signal reporter, and single-layer WS₂ is used as a quencher with a negatively charged surface. The ssDNA forms complexes with PFVCN due to much stronger electrostatic interactions between cationic PFVCN and anionic ssDNA, whereas PFVCN emits yellow fluorescence. When ssDNA is hydrolyzed by S1 nuclease or hydroxyl radicals into small fragments, the interactions between the fragmented DNA and PFVCN become weaker, resulting in PFVCN being adsorbed on the surface of WS₂ and the fluorescence being quenched through fluorescence resonance energy transfer. The new method based on PFVCN and WS₂ can sense S1 nuclease with a low detection limit of 5 × 10(-6) U/mL. Additionally, this method is cost-effective by using affordable WS₂ as an energy acceptor without the need for dye-labeled ssDNA. Furthermore, the method provides a new platform for the nuclease assay and reactive oxygen species, and provides promising applications for drug screening.

DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

2017-02-01

Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

Applicability of new degradable hypericin-polymer-conjugates as photosensitizers: principal mode of action demonstrated by in vitro models.

PubMed

Feinweber, Daniela; Verwanger, Thomas; Brüggemann, Oliver; Teasdale, Ian; Krammer, Barbara

2014-11-01

Two series of water soluble novel conjugates of the photosensitizer hypericin were prepared and evaluated for their use as agents for photodynamic therapy, with covalently and non-covalently loaded hypericin on functionalised, hydrolytically degradable inorganic-organic hybrid polyphosphazenes. The conjugates showed excellent aqueous solubility and similar fluorescence spectra to pristine hypericin. Detailed in vitro investigations revealed that the substances were non-toxic in the dark over a wide concentration range, but displayed phototoxicity upon irradiation. Cell uptake studies showed rapid uptake with localization of hypericin observed in endoplasmic reticulum, Golgi complex and particularly in the lysosomes. Furthermore, a DNA fragmentation assay revealed that the photosensitizer conjugates are efficient inducers of apoptosis with some tumor cell selectivity caused by faster and enhanced accumulation in A431 than in HaCaT cells, and thus a moderately higher phototoxicity of A431 compared to HaCaT cells. These novel photosensitizer conjugates hence represent viable hydrolytically degradable alternatives for the advanced delivery of hypericin.

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

PubMed

Mumcuoglu, Didem; Sardan Ekiz, Melis; Gunay, Gokhan; Tekinay, Turgay; Tekinay, Ayse B; Guler, Mustafa O

2016-05-11

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

Direct cell-cell contact activates SigM to express the ESX-4 secretion system in Mycobacterium smegmatis.

PubMed

Clark, Ryan R; Judd, Julius; Lasek-Nesselquist, Erica; Montgomery, Sarah A; Hoffmann, Jennifer G; Derbyshire, Keith M; Gray, Todd A

2018-06-25

Conjugal cell-cell contact between strains of Mycobacterium smegmatis induces the esxUT transcript, which encodes the putative primary substrates of the ESAT-6 secretion system 4 (ESX-4) secretion system. This recipient response was required for conjugal transfer of chromosomal DNA from the donor strain. Here we show that the extracytoplasmic σ factor, SigM, is a cell contact-dependent activator of ESX-4 expression and is required for conjugal transfer of DNA in the recipient strain. The SigM regulon includes genes outside the seven-gene core esx4 locus that we show are also required for conjugation, and we show that some of these SigM-induced proteins likely function through ESX-4. A fluorescent reporter revealed that SigM is specifically activated in recipient cells in direct contact with donor cells. Coculture RNA-seq experiments indicated that SigM regulon induction occurred early and before transconjugants are detected. This work supports a model wherein donor contact with the recipient cell surface inactivates the transmembrane anti-SigM, thereby releasing SigM. Free SigM induces an extended ESX-4 secretion system, resulting in changes that facilitate chromosomal transfer. The contact-dependent inactivation of an extracytoplasmic σ-factor that tightly controls ESX-4 activity suggests a mechanism dedicated to detect, and appropriately respond to, external stimuli from mycobacteria.

Stabilization of water in oil in water (W/O/W) emulsion using whey protein isolate-conjugated durian seed gum: enhancement of interfacial activity through conjugation process.

PubMed

Tabatabaee Amid, Bahareh; Mirhosseini, Hamed

2014-01-01

The present work was conducted to investigate the effect of purification and conjugation processes on functional properties of durian seed gum (DSG) used for stabilization of water in oil in water (W/O/W) emulsion. Whey protein isolate (WPI) was conjugated to durian seed gum through the covalent linkage. In order to prepare WPI-DSG conjugate, covalent linkage of whey protein isolate to durian seed gum was obtained by Maillard reaction induced by heating at 60 °C and 80% (±1%) relative humidity. SDS-polyacrylamide gel electrophoresis was used to test the formation of the covalent linkage between whey protein isolate and durian seed gum after conjugation process. In this study, W/O/W stabilized by WPI-conjugated DSG A showed the highest interface activity and lowest creaming layer among all prepared emulsions. This indicated that the partial conjugation of WPI to DSG significantly improved its functional characteristics in W/O/W emulsion. The addition of WPI-conjugated DSG to W/O/W emulsion increased the viscosity more than non-conjugated durian seed gum (or control). This might be due to possible increment of the molecular weight after linking the protein fraction to the structure of durian seed gum through the conjugation process. Copyright © 2013 Elsevier B.V. All rights reserved.

Energy-independent intracellular gene delivery mediated by polymeric biomimetics of cell-penetrating peptides.

PubMed

Chae, Su Young; Kim, Hyun June; Lee, Min Sang; Jang, Yeon Lim; Lee, Yuhan; Lee, Soo Hyeon; Lee, Kyuri; Kim, Sun Hwa; Kim, Hong Tae; Chi, Sang-Cheol; Park, Tae Gwan; Jeong, Ji Hoon

2011-09-09

Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

PubMed

Mukherjee, Anirban; Vasquez, Karen M

2011-08-01

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Importance of Dichloroacetate and Trichloroacetate to the Hepatocarcinogenic Response to Trichloroeylene in B6C3F1 Mice

DTIC Science & Technology

1989-10-15

Baker Co., polysorbate (Tween 80), beta- glucuronidase (Type VII), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), clofibrate , phenobllbital, DCA, TCA...TCOH conjugated wit’a glucuronic acid was determined in an aliquot of sample treated with beta-glucuronidase. A Varian Model 3700 gas chromatQgraph...diaminobenzoic acid fluorimetric assay. The fraction of DNA unwound during the two hour incubation at OC was calculated as: (Total DNA - DS DNA)t

Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

PubMed

Teo, Pei Yun; Yang, Chuan; Hedrick, James L; Engler, Amanda C; Coady, Daniel J; Ghaem-Maghami, Sadaf; George, Andrew J T; Yang, Yi Yan

2013-10-01

Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 μm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and ~100% substitution of primary amine groups leads to significantly lower gene transfection efficiency. These findings provide insights to modification of PEI for development of effective and non-cytotoxic non-viral vectors. Copyright © 2013 Elsevier Ltd. All rights reserved.

Probing the recognition surface of a DNA triplex: binding studies with intercalator-neomycin conjugates.

PubMed

Xue, Liang; Xi, Hongjuan; Kumar, Sunil; Gray, David; Davis, Erik; Hamilton, Paris; Skriba, Michael; Arya, Dev P

2010-07-06

Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA).2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalene diimide-neomycin (3) and anthraquinone-neomycin (4) conjugates were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2) conjugates, in our investigations. A combination of experiments, including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC), revealed that all four conjugates (1-4) stabilized poly(dA).2poly(dT) much more than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (T(m3-->2)) at which poly(dA).2poly(dT) dissociated into poly(dA).poly(dT) and poly(dT) increased dramatically (>12 degrees C) in the presence of intercalator-neomycin conjugates (1-4) even at a very low concentration (2 muM). In contrast to intercalator-neomycin conjugates, the increment of T(m3-->2) of poly(dA).2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin conjugates (1-4) to poly(dA).2poly(dT) was also confirmed by competition dialysis and a fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compounds 1-4 had slightly larger binding site size ( approximately 7-7.5) with poly(dA).2poly(dT) as compared to neomycin ( approximately 6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA).2poly(dT) were derived from an integrated van't Hoff equation using the T(m3-->2) values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA).2poly(dT) increased in the following order: neomycin < 1 < 3 < 4 < 2. Among them, the binding constant [(2.7 +/- 0.3) x 10(8) M(-1)] of 2 with poly(dA).2poly(dT) was the highest, almost 1000-fold greater than that of neomycin. The binding of compounds 1-4 with poly(dA).2poly(dT) was mostly enthalpy-driven and gave negative DeltaC(p) values. The results described here suggest that the binding affinity of intercalator-neomycin conjugates for poly(dA).2poly(dT) increases as a function of the surface area of the intercalator moiety.

A magnetic nanobead-based bioassay provides sensitive detection of single- and biplex bacterial DNA using a portable AC susceptometer.

PubMed

Strömberg, Mattias; Zardán Gómez de la Torre, Teresa; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria

2014-01-01

Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin-biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM-NDA further towards implementation in point-of-care and outpatient settings. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-License, which permits use and distribution in any medium, provided the original work is properly cited.

Multi-step high-throughput conjugation platform for the development of antibody-drug conjugates.

PubMed

Andris, Sebastian; Wendeler, Michaela; Wang, Xiangyang; Hubbuch, Jürgen

2018-07-20

Antibody-drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development. In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7 min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates. Copyright © 2018 Elsevier B.V. All rights reserved.

A DNA-Encapsulated and Fluorescent Ag 10 6+ Cluster with a Distinct Metal-Like Core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petty, Jeffrey T.; Ganguly, Mainak; Rankine, Ian J.

Silver cluster–DNA complexes are optical chromophores, and pairs of these conjugates can be toggled from fluorescently dim to bright states using DNA hybridization. This paper highlights spectral and structural differences for a specific cluster pair. We have previously characterized a cluster with low emission and violet absorption that forms a compact structure with single-stranded oligonucleotides. We now consider its counterpart with blue absorption and strong green emission. This cluster develops with a single-stranded/duplex DNA construct and is favored by low silver concentrations with ≲8 Ag+:DNA, an oxygen atmosphere, and neutral pH. The resulting cluster displays key signatures of a molecularmore » metal with well-defined absorption/emission bands at 490/550 nm, and with a fluorescence quantum yield of 15% and lifetime of 2.4 ns. The molecular cluster conjugates with the larger DNA host because it chromatographically elutes with the DNA and it exhibits circular dichroism. The silver cluster is identified as Ag106+ using two modes of mass spectrometry and elemental analysis. Our key finding is that it adopts a low-dimensional shape, as determined from a Ag K-edge extended X-ray absorption fine structure analysis. The Ag0 in this oxidized cluster segregates from the Ag+ via a sparse number of metal-like bonds and a denser network of silver–DNA bonds. This structure contrasts with the compact, octahedral-like shape of the violet counterpart to the blue cluster, which is also a Ag106+ species. We consider that the blue- and violet-absorbing clusters may be isomers with shapes that are controlled by the secondary structures of their DNA templates.« less

Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure

PubMed Central

Guillonneau, F.; Guieysse, A. L.; Nocentini, S.; Giovannangeli, C.; Praseuth, D.

2004-01-01

Targeting DNA-damaging agents to specific DNA sites by using sequence-specific DNA ligands has been successful in directing genomic modifications. The understanding of repair processing of such targeted damage and the influence of the adjacent complex is largely unknown. In this way, directed interstrand cross-links (ICLs) have already been generated by psoralen targeting. The mechanisms responsible for ICL removal are far from being understood in mammalian cells, with the proposed involvement of both mutagenic and recombinogenic pathways. Here, a unique ICL was introduced at a selected site by photoactivation of a psoralen moiety with the use of psoralen conjugates of triplex-forming oligonucleotides. The processing of psoralen ICL was evaluated in vitro and in cells for two types of cross-linked substrates, either containing a psoralen ICL alone or with an adjacent triple-stranded structure. We show that the presence of a neighbouring triplex structure interferes with different stages of psoralen ICL processing: (i) the ICL-induced DNA repair synthesis in HeLa cell extracts is inhibited by the triplex structure, as measured by the efficiency of ‘true’ and futile repair synthesis, stopping at the ICL site; (ii) in HeLa cells, the ICL removal via a nucleotide excision repair (NER) pathway is delayed in the presence of a neighbouring triplex; and (iii) the binding to ICL of recombinant xeroderma pigmentosum A protein, which is involved in pre-incision recruitment of NER factors is impaired by the presence of the third DNA strand. These data characterize triplex-induced modulation of ICL repair pathways at specific steps, which might have implications for the controlled induction of targeted genomic modifications and for the associated cellular responses. PMID:14966263

DNA-Based Applications in Nanobiotechnology

PubMed Central

Abu-Salah, Khalid M.; Ansari, Anees A.; Alrokayan, Salman A.

2010-01-01

Biological molecules such as deoxyribonucleic acid (DNA) have shown great potential in fabrication and construction of nanostructures and devices. The very properties that make DNA so effective as genetic material also make it a very suitable molecule for programmed self-assembly. The use of DNA to assemble metals or semiconducting particles has been extended to construct metallic nanowires and functionalized nanotubes. This paper highlights some important aspects of conjugating the unique physical properties of dots or wires with the remarkable recognition capabilities of DNA which could lead to miniaturizing biological electronics and optical devices, including biosensors and probes. Attempts to use DNA-based nanocarriers for gene delivery are discussed. In addition, the ecological advantages and risks of nanotechnology including DNA-based nanobiotechnology are evaluated. PMID:20652049

DNA-based applications in nanobiotechnology.

PubMed

Abu-Salah, Khalid M; Ansari, Anees A; Alrokayan, Salman A

2010-01-01

Biological molecules such as deoxyribonucleic acid (DNA) have shown great potential in fabrication and construction of nanostructures and devices. The very properties that make DNA so effective as genetic material also make it a very suitable molecule for programmed self-assembly. The use of DNA to assemble metals or semiconducting particles has been extended to construct metallic nanowires and functionalized nanotubes. This paper highlights some important aspects of conjugating the unique physical properties of dots or wires with the remarkable recognition capabilities of DNA which could lead to miniaturizing biological electronics and optical devices, including biosensors and probes. Attempts to use DNA-based nanocarriers for gene delivery are discussed. In addition, the ecological advantages and risks of nanotechnology including DNA-based nanobiotechnology are evaluated.

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs.

PubMed

Vargas, Eva; Torrente-Rodríguez, Rebeca M; Ruiz-Valdepeñas Montiel, Víctor; Povedano, Eloy; Pedrero, María; Montoya, Juan J; Campuzano, Susana; Pingarrón, José M

2017-11-09

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA-RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H₂O₂/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at -0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA-RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNA t ) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.

Adsorption of doxorubicin on citrate-capped gold nanoparticles: insights into engineering potent chemotherapeutic delivery systems

NASA Astrophysics Data System (ADS)

Curry, Dennis; Cameron, Amanda; MacDonald, Bruce; Nganou, Collins; Scheller, Hope; Marsh, James; Beale, Stefanie; Lu, Mingsheng; Shan, Zhi; Kaliaperumal, Rajendran; Xu, Heping; Servos, Mark; Bennett, Craig; Macquarrie, Stephanie; Oakes, Ken D.; Mkandawire, Martin; Zhang, Xu

2015-11-01

Gold nanomaterials have received great interest for their use in cancer theranostic applications over the past two decades. Many gold nanoparticle-based drug delivery system designs rely on adsorbed ligands such as DNA or cleavable linkers to load therapeutic cargo. The heightened research interest was recently demonstrated in the simple design of nanoparticle-drug conjugates wherein drug molecules are directly adsorbed onto the as-synthesized nanoparticle surface. The potent chemotherapeutic, doxorubicin often serves as a model drug for gold nanoparticle-based delivery platforms; however, the specific interaction facilitating adsorption in this system remains understudied. Here, for the first time, we propose empirical and theoretical evidence suggestive of the main adsorption process where (1) hydrophobic forces drive doxorubicin towards the gold nanoparticle surface before (2) cation-π interactions and gold-carbonyl coordination between the drug molecule and the cations on AuNP surface facilitate DOX adsorption. In addition, biologically relevant compounds, such as serum albumin and glutathione, were shown to enhance desorption of loaded drug molecules from AuNP at physiologically relevant concentrations, providing insight into the drug release and in vivo stability of such drug conjugates.Gold nanomaterials have received great interest for their use in cancer theranostic applications over the past two decades. Many gold nanoparticle-based drug delivery system designs rely on adsorbed ligands such as DNA or cleavable linkers to load therapeutic cargo. The heightened research interest was recently demonstrated in the simple design of nanoparticle-drug conjugates wherein drug molecules are directly adsorbed onto the as-synthesized nanoparticle surface. The potent chemotherapeutic, doxorubicin often serves as a model drug for gold nanoparticle-based delivery platforms; however, the specific interaction facilitating adsorption in this system remains understudied. Here, for the first time, we propose empirical and theoretical evidence suggestive of the main adsorption process where (1) hydrophobic forces drive doxorubicin towards the gold nanoparticle surface before (2) cation-π interactions and gold-carbonyl coordination between the drug molecule and the cations on AuNP surface facilitate DOX adsorption. In addition, biologically relevant compounds, such as serum albumin and glutathione, were shown to enhance desorption of loaded drug molecules from AuNP at physiologically relevant concentrations, providing insight into the drug release and in vivo stability of such drug conjugates. Electronic supplementary information (ESI) available: DOX-AuNP absorption spectra and colored solution images, citrate displacement data, original DOX-AuNP loading isotherm, XPS data and TEM micrographs, modelling data. See DOI: 10.1039/c5nr05826k

Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification.

PubMed

Tang, Juan; Huang, Yapei; Liu, Huiqiong; Zhang, Cengceng; Tang, Dianping

2016-12-01

A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) in foodstuff. The system consisted of anti-AFB 1 antibody labeled DNA 1 (Ab-DNA 1 ), AFB 1 -bovine serum albumin (BSA)-conjugated DNA 2 (AFB 1 -DNA 2 ), and methylene blue functionalized hairpin DNA. Owing to a specific antigen-antibody reaction between anti-AFB 1 and AFB 1 -BSA, the immunocomplex formed assisted the proximity hybridization of DNA 1 with DNA 2 , thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB 1 , the analyte competed with AFB 1 -DNA 2 for the conjugated anti-AFB 1 on the Ab-DNA 1 , subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB 1 in a dynamic working range of 0.01-30 ng mL -1 with a detection limit of 4.8 pg mL -1 . In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB 1 -spiked peanut samples, giving results that matched well with those obtained with a commercial AFB 1 ELISA kit.

Improving Nucleoside Analogs via Lipid Conjugation; Is fatter any better?

PubMed Central

Alexander, Peter; Kucera, Gregory; Pardee, Timothy S.

2016-01-01

In the past few decades, nucleoside analog drugs have been used to treat a large variety of cancers. These antimetabolite drugs mimic nucleosides and interfere with chain lengthening upon incorporation into the DNA or RNA of actively replicating cells. However, efficient delivery of these drugs is limited due to their pharmacokinetic properties, and tumors often develop drug resistance. In addition, nucleoside analogs are generally hydrophilic, resulting in poor bioavailability and impaired blood-brain barrier penetration. Conjugating these drugs to lipids modifies their pharmacokinetic properties and may improve in vivo efficacy. This review will cover recent advances in the field of conjugation of phospholipids to nucleoside analogs. This includes conjugation of myristic acid, 12-thioethyldodecanoic acid, 5-elaidic acid esters, phosphoramidate, and self-emulsifying formulations. Relevant in vitro and in vivo data will be discussed for each drug, as well as any available data from clinical trials. PMID:26829896

From oleic acid-capped iron oxide nanoparticles to polyethyleneimine-coated single-particle magnetofectins

NASA Astrophysics Data System (ADS)

Cruz-Acuña, Melissa; Maldonado-Camargo, Lorena; Dobson, Jon; Rinaldi, Carlos

2016-09-01

Various inorganic nanoparticle designs have been developed and used as non-viral gene carriers. Magnetic gene carriers containing polyethyleneimine (PEI), a well-known transfection agent, have been shown to improve DNA transfection speed and efficiency in the presence of applied magnetic field gradients that promote particle-cell interactions. Here we report a method to prepare iron oxide nanoparticles conjugated with PEI that: preserves the narrow size distribution of the nanoparticles, conserves magnetic properties throughout the process, and results in efficient transfection. We demonstrate the ability of the particles to electrostatically bind with DNA and transfect human cervical cancer (HeLa) cells by the use of an oscillating magnet array. Their transfection efficiency is similar to that of Lipofectamine 2000™, a commercial transfection reagent. PEI-coated particles were subjected to acidification, and acidification in the presence of salts, before DNA binding. Results show that although these pre-treatments did not affect the ability of particles to bind DNA they did significantly enhanced transfection efficiency. Finally, we show that these magnetofectins (PEI-MNP/DNA) complexes have no effect on the viability of cells at the concentrations used in the study. The systematic preparation of magnetic vectors with uniform physical and magnetic properties is critical to progressing this non-viral transfection technology.

CHEMICAL MODIFICATION MODULATES ESTROGENIC ACTIVITY, OXIDATIVE REACTIVITY, & METABOLIC STABILITY IN 4′F-DMA, A NEW BENZOTHIOPHENE SELECTIVE ESTROGEN RECEPTOR MODULATOR

PubMed Central

Liu, Hong; Bolton, Judy L.; Thatcher, Gregory R. J.

2008-01-01

The benzothiophene SERMs raloxifene and arzoxifene, in the clinic or clinical trials for treatment of breast cancer and postmenopausal symptoms, are highly susceptible to oxidative metabolism and formation of electrophilic metabolites. 4′F-DMA, fluoro-substituted desmethyl arzoxifene (DMA), showed attenuated oxidation to quinoids in incubation with rat hepatocytes as well as in rat and human liver microsomes. Incubations of 4′F-DMA with hepatocytes yielded only one glucuronide conjugate and no GSH conjugates; whereas DMA underwent greater metabolism giving two glucuronide conjugates, one sulfate conjugate, and two GSH conjugates. Phase I and phase II metabolism was further evaluated in human small intestine microsomes and in human intestinal Caco-2 cells. In comparison to DMA, 4′F-DMA formed significantly less glucuronide and sulfate conjugates. The formation of quinoids was futher explored in hepatocytes in which DMA was observed to give concentration and time dependent depletion of GSH accompanied by damage to DNA which showed inverse dependence on GSH; in contrast, GSH depletion and DNA damage were almost completely abrogated in incubations with 4′F-DMA. 4′F-DMA shows ligand binding affinity to ERα and ERβ with similarity to both raloxifene and to DMA. ER-mediated biological activity was measured with the ERE-luciferase reporter system in transfected MCF-7 cells and Ishikawa cells, and in MCF-7 cells proliferation was measured. In all systems, 4′F-DMA exhibited anitestrogenic acitivty of comparable potency to raloxifene, but did not manifest estrogenic properties, mirroring previous results on inhibition of estradiol-mediated induction of alkaline phosphatase activity in Ishikawa cells. These results suggest that 4′F-DMA might be an improved benzothiophene SERM with similar antiestrogenic activity to raloxifene, but improved metabolic stability and attenuated toxicity; showing that simple chemical modification can abrogate oxidative bioactivation to potentially toxic metabolites without loss of activity. PMID:16780356

Electrical and Electron-Phonon Interactions in Graphene-Based Nanostructures and Aptamer-Based Electrical Sensors

NASA Astrophysics Data System (ADS)

Qian, Jun

This research work contains two main parts: the theoretical study of confined phonon modes and electron states in confined graphene nanostructures; the experimental part including two topics about fabricating a graphene-FET aptamer-sensor for cocaine detection and the study of the electronic transport properties of dsDNA. In the theory part, we study the confined optical phonon modes in graphene nanoribbons (GNR) and rectangular graphene quantum dots (RGQD) by the elastic continuum model. The carrier states are studied by effective mass approximation. The phonon bottleneck effect is expected in general for RGQDs. The scattering rates are calculated for specific RGQDs with carefully chosen dimensions to fulfill the momentum and energy conservation conditions. In the experimental part, we have developed a combined technique of semiconductor processes and molecular biological protocols to fabricate a signal-off graphene-FET aptamer-sensor for cocaine. In addition, DNA transport properties were studied by STM on GNP-dsDNA-Au conjugates in atmospheric condition. The dsDNA-complexes exhibit as a slightly n-type semiconductor by simulated with a Landauer-type model. A geometrical model is proposed to explain the distinct I-V spectra.

Androgen Receptor Antagonism By Divalent Ethisterone Conjugates In Castrate-Resistant Prostate Cancer Cells

PubMed Central

Levine, Paul M.; Lee, Eugine; Greenfield, Alex; Bonneau, Richard; Logan, Susan K.; Garabedian, Michael J.; Kirshenbaum, Kent

2013-01-01

Sustained treatment of prostate cancer with Androgen Receptor (AR) antagonists can evoke drug resistance, leading to castrate-resistant disease. Elevated activity of the AR is often associated with this highly aggressive disease state. Therefore, new therapeutic regimens that target and modulate AR activity could prove beneficial. We previously introduced a versatile chemical platform to generate competitive and non-competitive multivalent peptoid oligomer conjugates that modulate AR activity. In particular, we identified a linear and a cyclic divalent ethisterone conjugate that exhibit potent anti-proliferative properties in LNCaP-abl cells, a model of castrate-resistant prostate cancer. Here, we characterize the mechanism of action of these compounds utilizing confocal microscopy, time-resolved fluorescence resonance energy transfer, chromatin immunoprecipitation, flow cytometry, and microarray analysis. The linear conjugate competitively blocks AR action by inhibiting DNA binding. In addition, the linear conjugate does not promote AR nuclear localization or co-activator binding. In contrast, the cyclic conjugate promotes AR nuclear localization and induces cell-cycle arrest, despite its inability to compete against endogenous ligand for binding to AR in vitro. Genome-wide expression analysis reveals that gene transcripts are differentially affected by treatment with the linear or cyclic conjugate. Although the divalent ethisterone conjugates share extensive chemical similarities, we illustrate that they can antagonize the AR via distinct mechanisms of action, establishing new therapeutic strategies for potential applications in AR pharmacology. PMID:22871957

Integrative and conjugative elements and their hosts: composition, distribution and organization.

PubMed

Cury, Jean; Touchon, Marie; Rocha, Eduardo P C

2017-09-06

Conjugation of single-stranded DNA drives horizontal gene transfer between bacteria and was widely studied in conjugative plasmids. The organization and function of integrative and conjugative elements (ICE), even if they are more abundant, was only studied in a few model systems. Comparative genomics of ICE has been precluded by the difficulty in finding and delimiting these elements. Here, we present the results of a method that circumvents these problems by requiring only the identification of the conjugation genes and the species' pan-genome. We delimited 200 ICEs and this allowed the first large-scale characterization of these elements. We quantified the presence in ICEs of a wide set of functions associated with the biology of mobile genetic elements, including some that are typically associated with plasmids, such as partition and replication. Protein sequence similarity networks and phylogenetic analyses revealed that ICEs are structured in functional modules. Integrases and conjugation systems have different evolutionary histories, even if the gene repertoires of ICEs can be grouped in function of conjugation types. Our characterization of the composition and organization of ICEs paves the way for future functional and evolutionary analyses of their cargo genes, composed of a majority of unknown function genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

GLUT1-mediated selective tumor targeting with fluorine containing platinum(II) glycoconjugates

PubMed Central

Liu, Ran; Fu, Zheng; Zhao, Meng; Gao, Xiangqian; Li, Hong; Mi, Qian; Liu, Pengxing; Yang, Jinna; Yao, Zhi; Gao, Qingzhi

2017-01-01

Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design. PMID:28467806

GLUT1-mediated selective tumor targeting with fluorine containing platinum(II) glycoconjugates.

PubMed

Liu, Ran; Fu, Zheng; Zhao, Meng; Gao, Xiangqian; Li, Hong; Mi, Qian; Liu, Pengxing; Yang, Jinna; Yao, Zhi; Gao, Qingzhi

2017-06-13

Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design.

Polyethyleneimine-lipid conjugate-based pH-sensitive micellar carrier for gene delivery

PubMed Central

Sawant, Rupa R.; Sriraman, Shravan Kumar; Navarro, Gemma; Biswas, Swati; Dalvi, Riddhi A.; Torchilin, Vladimir P.

2012-01-01

A low molecular weight polyethyleneimine (PEI 1.8 kDa) was modified with dioleoylphosphatidylethanolamine (PE) to form the PEI-PE conjugate investigated as a transfection vector. The optimized PEI-PE/pDNA complexes at an N/P ratio of 16 had a particle size of 225 nm, a surface charge of +31 mV, and protected the pDNA from the action of DNase I. The PEI-PE conjugate had a critical micelle concentration (CMC) of about 34 μg/ml and exhibited no toxicity compared to a high molecular weight PEI (PEI 25 kDa) as tested with B16-F10 melanoma cells. The B16-F10 cells transfected with PEI-PE/pEGFP complexes showed protein expression levels higher than with PEI-1.8 or PEI-25 vectors. Complexes prepared with YOYO 1-labeled pEGFP confirmed the enhanced delivery of the plasmid with PEI-PE compared to PEI-1.8 and PEI-25. The PEI-PE/pDNA complexes were also mixed with various amounts of micelle-forming material, polyethylene glycol (PEG)-PE to improve biocompatibility. The resulting particles exhibited a neutral surface charge, resistance to salt-induced aggregation, and good transfection activity in the presence of serum in complete media. The use of the low-pH-degradable PEG-hydrazone-PE produced particles with transfection activity sensitive to changes in pH consistent with the relatively acidic tumor environment. PMID:22365809

Morphological priming by itself: a study of Portuguese conjugations.

PubMed

Veríssimo, João; Clahsen, Harald

2009-07-01

Does the language processing system make use of abstract grammatical categories and representations that are not directly visible from the surface form of a linguistic expression? This study examines stem-formation processes and conjugation classes, a case of 'pure' morphology that provides insight into the role of grammatical structure in language processing. We report results from a cross-modal priming experiment examining 1st and 3rd conjugation verb forms in Portuguese. Although items were closely matched with respect to a range of non-morphological factors, distinct priming patterns were found for 1st and 3rd conjugation stems. We attribute the observed priming patterns to different representations of conjugational stems, combinatorial morphologically structured ones for 1st conjugation and un-analyzed morphologically unstructured ones for 3rd conjugation stems. Our findings underline the importance of morphology for language comprehension indicating that morphological analysis goes beyond the identification of grammatical morphemes.

Synergetic approach for simple and rapid conjugation of gold nanoparticles with oligonucleotides.

PubMed

Li, Jiuxing; Zhu, Binqing; Yao, Xiujie; Zhang, Yicong; Zhu, Zhi; Tu, Song; Jia, Shasha; Liu, Rudi; Kang, Huaizhi; Yang, Chaoyong James

2014-10-08

Attaching thiolated DNA on gold nanoparticles (AuNPs) has been extremely important in nanobiotechnology because DNA-AuNPs combine the programmability and molecular recognition properties of the biopolymers with the optical, thermal, and catalytic properties of the inorganic nanomaterials. However, current standard protocols to attach thiolated DNA on AuNPs involve time-consuming, tedious steps and do not perform well for large AuNPs, thereby greatly restricting applications of DNA-AuNPs. Here we demonstrate a rapid and facile strategy to attach thiolated DNA on AuNPs based on the excellent stabilization effect of mPEG-SH on AuNPs. AuNPs are first protected by mPEG-SH in the presence of Tween 20, which results in excellent stability of AuNPs in high ionic strength environments and extreme pHs. A high concentration of NaCl can be applied to the mixture of DNA and AuNP directly, allowing highly efficient DNA attachment to the AuNP surface by minimizing electrostatic repulsion. The entire DNA loading process can be completed in 1.5 h with only a few simple steps. DNA-loaded AuNPs are stable for more than 2 weeks at room temperature, and they can precisely hybridize with the complementary sequence, which was applied to prepare core-satellite nanostructures. Moreover, cytotoxicity assay confirmed that the DNA-AuNPs synthesized by this method exhibit lower cytotoxicity than those prepared by current standard methods. The proposed method provides a new way to stabilize AuNPs for rapid and facile loading thiolated DNA on AuNPs and will find wide applications in many areas requiring DNA-AuNPs, including diagnosis, therapy, and imaging.

Toward efficient Zn(II)-based artificial nucleases.

PubMed

Boseggia, Elisa; Gatos, Maddalena; Lucatello, Lorena; Mancin, Fabrizio; Moro, Stefano; Palumbo, Manlio; Sissi, Claudia; Tecilla, Paolo; Tonellato, Umberto; Zagotto, Giuseppe

2004-04-14

A series of cis-cis-triaminocyclohexane Zn(II) complex-anthraquinone intercalator conjugates, designed in such a way to allow their easy synthesis and modification, have been investigated as hydrolytic cleaving agents for plasmid DNA. The ligand structure comprises a triaminocyclohexane platform linked by means of alkyl spacers of different length (from C(4) to C(8)) to the anthraquinone group which may intercalate the DNA. At a concentration of 5 microM, the complex of the derivative with a C(8) alkyl spacer induces the hydrolytic stand scission of supercoiled DNA with a rate of 4.6 x 10(-6) s(-1) at pH 7 and 37 degrees C. The conjugation of the metal complex with the anthraquinone group leads to a 15-fold increase of the cleavage efficiency when compared with the anthraquinone lacking Zn-triaminocyclohexane complex. The straightforward synthetic procedure employed, allowing a systematic change of the spacer length, made possible to gain more insight on the role of the intercalating group in determining the reactivity of the systems. Comparison of the reactivity of the different complexes shows a remarkable increase of the DNA cleaving efficiency with the length of the spacer. In the case of too-short spacers, the advantages due to the increased DNA affinity are canceled due to the incorrect positioning of the reactive group, thus leading to cleavage inhibition.

Food isolate Listeria monocytogenes harboring tetM gene plasmid-mediated exchangeable to Enterococcus faecalis on the surface of processed cheese.

PubMed

Haubert, Louise; Cunha, Carlos Eduardo Pouey da; Lopes, Graciela Völz; Silva, Wladimir Padilha da

2018-05-01

The genetic basis of tetracycline resistance in a food isolate Listeria monocytogenes (Lm16) was evaluated. Resistance to tetracycline was associated with the presence of the tetM gene in plasmid DNA. The sequence of tetM showed 100% of similarity with the Enterococcus faecalis sequences found in the EMBL database, suggesting that Lm16 received this gene from E. faecalis. Various size bands were detected in the DNA plasmid analysis, the largest being approximately 54.38 kb. Transferability of the tetM gene was achieved in vitro by agar matings between Lm16 and E. faecalis JH2-2, proving the potential for the spread of tetM by horizontal gene transfer. Furthermore, the conjugation experiments were performed on the surface of processed cheese, confirming the transferability in a food matrix. PCR assays were used to confirm the identity of E. faecalis and to detect the tetM gene in transconjugant bacteria. Additionally, the minimal inhibitory concentration for tetracycline and rifampicin and plasmid profiling were performed. This is the first report of a food isolate L. monocytogenes carrying the tetM gene in plasmid DNA, and it highlights the potential risk of spreading antimicrobial resistance genes between different bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

PubMed Central

Ramirez, Lisa S.; Wang, Jun

2016-01-01

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

Multi-colored fibers by self-assembly of DNA, histone proteins, and cationic conjugated polymers.

PubMed

Wang, Fengyan; Liu, Zhang; Wang, Bing; Feng, Liheng; Liu, Libing; Lv, Fengting; Wang, Yilin; Wang, Shu

2014-01-07

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color-encoded IPC fibers were also obtained based on the co-assembly of DNA, histone proteins, and blue-, green-, or red- (RGB-) emissive CCPs by tuning the fluorescence resonance energy-transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP-coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi-colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.

PubMed

Liu, Xingfen; Ouyang, Lan; Cai, Xiaohui; Huang, Yanqin; Feng, Xiaomiao; Fan, Quli; Huang, Wei

2013-03-15

Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and "turn-on" detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1 pg/μL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease

PubMed Central

Nelson, Margaret-Ann M; Baba, Shahid P; Andersonc, Ethan J

2017-01-01

Aldehydes are continuously formed in biological systems through enzyme-dependent and spontaneous oxidation of lipids, glucose, and primary amines. These highly reactive, biogenic electrophiles can become toxic via covalent modification of proteins, lipids and DNA. Thus, agents that scavenge aldehydes through conjugation have therapeutic value for a number of major cardiovascular diseases. Several commonly-prescribed drugs (e.g., hydralazine) have been shown to have potent aldehyde-conjugating properties which may contribute to their beneficial effects. Herein, we briefly describe the major sources and toxicities of biogenic aldehydes in cardiovascular system, and provide an overview of drugs that are known to have aldehyde-conjugating effects. Some compounds of phytochemical origin, and histidyl-dipeptides with emerging therapeutic value in this area are also discussed. PMID:28528297

Precision polymers and 3D DNA nanostructures: emergent assemblies from new parameter space.

PubMed

Serpell, Christopher J; Edwardson, Thomas G W; Chidchob, Pongphak; Carneiro, Karina M M; Sleiman, Hanadi F

2014-11-05

Polymer self-assembly and DNA nanotechnology have both proved to be powerful nanoscale techniques. To date, most attempts to merge the fields have been limited to placing linear DNA segments within a polydisperse block copolymer. Here we show that, by using hydrophobic polymers of a precisely predetermined length conjugated to DNA strands, and addressable 3D DNA prisms, we are able to effect the formation of unprecedented monodisperse quantized superstructures. The structure and properties of larger micelles-of-prisms were probed in depth, revealing their ability to participate in controlled release of their constituent nanostructures, and template light-harvesting energy transfer cascades, mediated through both the addressability of DNA and the controlled aggregation of the polymers.

Inhibition of DNA synthesis in cultured lymphocytes and tumor cells by extracts of betel nut, tobacco, and miang leaf, plant substances associated with cancer of the ororespiratory epithelium.

PubMed

Yang, J A; Huber, S A; Lucas, Z J

1979-12-01

The high incidence of oropharyngeal, esophageal, and laryngeal cancers in certain parts of the world has been ascribed to conjugated tannins found in certain folk medicinal herbs. We extracted miang leaf and betel nut with phosphate-buffered saline (0.14 M NaCl, 0.15 M potassium phosphate buffer, pH 7.4) and found that the extracts inhibited [3H]thymidine incorporation by phytohemagglutinin-stimulated human lymphocytes and by rat mammary tumor and mouse L-cells in logarithmic growth. Pretreating the lymphocytes for 1 or 4 hr with the extracts inhibited phytohemagglutinin-induced thymidine incorporation 72 hr later. At concentrations of 2.5 volumes % or lower, miang and betel nut extracts inhibited thymidine incorporation by 40 to 98% without any apparent signs of toxicity as demonstrated by the 66Rb equilibrium assay. In addition, neither extract inhibited cytotoxicity of rat mammary tumor cells by immune syngeneic spleen cells. The molecular weights of the inhibitory factors were between 1,000 and 10,000 daltons as determined by ultrafiltration and were unaffected by boiling for 3 min or by treatment with alcohol and, therefore, are probably not proteins. This in vitro demonstration of inhibition of DNA synthesis by these plant extracts presumably enriched for conjugated tannins may relate to inhibition of growth of rats and chicks fed conjugated tanin-contaminated sorghum feed. The carcinogenic potential of either these extracts or conjugated tannins is not yet established.

Reversible Redox Activity by Ion-pH Dually Modulated Duplex Formation of i-Motif DNA with Complementary G-DNA.

PubMed

Chang, Soyoung; Kilic, Tugba; Lee, Chang Kee; Avci, Huseyin; Bae, Hojae; Oskui, Shirin Mesbah; Jung, Sung Mi; Shin, Su Ryon; Kim, Seon Jeong

2018-04-08

The unique biological features of supramolecular DNA have led to an increasing interest in biomedical applications such as biosensors. We have developed an i-motif and G-rich DNA conjugated single-walled carbon nanotube hybrid materials, which shows reversible conformational switching upon external stimuli such as pH (5 and 8) and presence of ions (Li⁺ and K⁺). We observed reversible electrochemical redox activity upon external stimuli in a quick and robust manner. Given the ease and the robustness of this method, we believe that pH- and ion-driven reversible DNA structure transformations will be utilized for future applications for developing novel biosensors.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

PubMed

Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

2015-05-12

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

Bacterial sex in dental plaque.

PubMed

Olsen, Ingar; Tribble, Gena D; Fiehn, Nils-Erik; Wang, Bing-Yan

2013-01-01

Genes are transferred between bacteria in dental plaque by transduction, conjugation, and transformation. Membrane vesicles can also provide a mechanism for horizontal gene transfer. DNA transfer is considered bacterial sex, but the transfer is not parallel to processes that we associate with sex in higher organisms. Several examples of bacterial gene transfer in the oral cavity are given in this review. How frequently this occurs in dental plaque is not clear, but evidence suggests that it affects a number of the major genera present. It has been estimated that new sequences in genomes established through horizontal gene transfer can constitute up to 30% of bacterial genomes. Gene transfer can be both inter- and intrageneric, and it can also affect transient organisms. The transferred DNA can be integrated or recombined in the recipient's chromosome or remain as an extrachromosomal inheritable element. This can make dental plaque a reservoir for antimicrobial resistance genes. The ability to transfer DNA is important for bacteria, making them better adapted to the harsh environment of the human mouth, and promoting their survival, virulence, and pathogenicity.

Polymer therapeutics: concepts and applications.

PubMed

Haag, Rainer; Kratz, Felix

2006-02-13

Polymer therapeutics encompass polymer-protein conjugates, drug-polymer conjugates, and supramolecular drug-delivery systems. Numerous polymer-protein conjugates with improved stability and pharmacokinetic properties have been developed, for example, by anchoring enzymes or biologically relevant proteins to polyethylene glycol components (PEGylation). Several polymer-protein conjugates have received market approval, for example the PEGylated form of adenosine deaminase. Coupling low-molecular-weight anticancer drugs to high-molecular-weight polymers through a cleavable linker is an effective method for improving the therapeutic index of clinically established agents, and the first candidates have been evaluated in clinical trials, including, N-(2-hydroxypropyl)methacrylamide conjugates of doxorubicin, camptothecin, paclitaxel, and platinum(II) complexes. Another class of polymer therapeutics are drug-delivery systems based on well-defined multivalent and dendritic polymers. These include polyanionic polymers for the inhibition of virus attachment, polycationic complexes with DNA or RNA (polyplexes), and dendritic core-shell architectures for the encapsulation of drugs. In this Review an overview of polymer therapeutics is presented with a focus on concepts and examples that characterize the salient features of the drug-delivery systems.

Recent Advances in Conjugated Polymer Materials for Disease Diagnosis.

PubMed

Lv, Fengting; Qiu, Tian; Liu, Libing; Ying, Jianming; Wang, Shu

2016-02-10

The extraordinary optical amplification and light-harvesting properties of conjugated polymers impart sensing systems with higher sensitivity, which meets the primary demands of early cancer diagnosis. Recent advances in the detection of DNA methylation and mutation with polyfluorene derivatives based fluorescence resonance energy transfer (FRET) as a means to modulate fluorescent responses attest to the great promise of conjugated polymers as powerful tools for the clinical diagnosis of diseases. To facilitate the ever-changing needs of diagnosis, the development of detection approaches and FRET signal analysis are highlighted in this review. Due to their exceptional brightness, excellent photostability, and low or absent toxicity, conjugated polymers are verified as superior materials for in-vivo imaging, and provide feasibility for future clinical molecular-imaging applications. The integration of conjugated polymers with clinical research has shown profound effects on diagnosis for the early detection of disease-related biomarkers, as well as in-vivo imaging, which leads to a multidisciplinary scientific field with perspectives in both basic research and application issues. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

PubMed

Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene

2018-01-01

Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random

PubMed Central

Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene

2018-01-01

Abstract Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world’s oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a “headful” type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated. PMID:29325123

Comparison of anti-EGFR-Fab’ conjugated immunoliposomes modified with two different conjugation linkers for siRNa delivery in SMMC-7721 cells

PubMed Central

Deng, Li; Zhang, Yingying; Ma, Lulu; Jing, Xiaolong; Ke, Xingfa; Lian, Jianhao; Zhao, Qiang; Yan, Bo; Zhang, Jinfeng; Yao, Jianzhong; Chen, Jianming

2013-01-01

Background Targeted liposome-polycation-DNA complex (LPD), mainly conjugated with antibodies using functionalized PEG derivatives, is an effective nanovector for systemic delivery of small interference RNA (siRNA). However, there are few studies reporting the effect of different conjugation linkers on LPD for gene silencing. To clarify the influence of antibody conjugation linkers on LPD, we prepared two different immunoliposomes to deliver siRNA in which DSPE-PEG-COOH and DSPE-PEG-MAL, the commonly used PEG derivative linkers, were used to conjugate anti-EGFR Fab’ with the liposome. Methods First, 600 μg of anti-EGFR Fab’ was conjugated with 28.35 μL of a micelle solution containing DSPE-PEG-MAL or DSPE-PEG-COOH, and then post inserted into the prepared LPD. Various liposome parameters, including particle size, zeta potential, stability, and encapsulation efficiency were evaluated, and the targeting ability and gene silencing activity of TLPD-FPC (DSPE-PEG-COOH conjugated with Fab’) was compared with that of TLPD-FPM (DSPE-PEG-MAL conjugated with Fab’) in SMMC-7721 hepatocellular carcinoma cells. Results There was no significant difference in particle size between the two TLPDs, but the zeta potential was significantly different. Further, although there was no significant difference in siRNA encapsulation efficiency, cell viability, or serum stability between TLPD-FPM and TLPD-FPC, cellular uptake of TLPD-FPM was significantly greater than that of TLPD-FPC in EGFR-overexpressing SMMC-7721 cells. The luciferase gene silencing efficiency of TLPD-FPM was approximately three-fold high than that of TLPD-FPC. Conclusion Different conjugation linkers whereby antibodies are conjugated with LPD can affect the physicochemical properties of LPD and antibody conjugation efficiency, thus directly affecting the gene silencing effect of TLPD. Immunoliposomes prepared by DSPE-PEG-MAL conjugation with anti-EGFR Fab’ are more effective than TLPD containing DSPE-PEG-COOH in targeting hepatocellular carcinoma cells for siRNA delivery. PMID:24023515

Internal-Modified Dithiol DNA–Directed Au Nanoassemblies: Geometrically Controlled Self–Assembly and Quantitative Surface–Enhanced Raman Scattering Properties

PubMed Central

Yan, Yuan; Shan, Hangyong; Li, Min; Chen, Shu; Liu, Jianyu; Cheng, Yanfang; Ye, Cui; Yang, Zhilin; Lai, Xuandi; Hu, Jianqiang

2015-01-01

In this work, a hierarchical DNA–directed self–assembly strategy to construct structure–controlled Au nanoassemblies (NAs) has been demonstrated by conjugating Au nanoparticles (NPs) with internal–modified dithiol single-strand DNA (ssDNA) (Au–B–A or A–B–Au–B–A). It is found that the dithiol–ssDNA–modified Au NPs and molecule quantity of thiol–modified ssDNA grafted to Au NPs play critical roles in the assembly of geometrically controlled Au NAs. Through matching Au–DNA self–assembly units, geometrical structures of the Au NAs can be tailored from one–dimensional (1D) to quasi–2D and 2D. Au–B–A conjugates readily give 1D and quasi–2D Au NAs while 2D Au NAs can be formed by A–B–Au–B–A building blocks. Surface-enhanced Raman scattering (SERS) measurements and 3D finite–difference time domain (3D-FDTD) calculation results indicate that the geometrically controllable Au NAs have regular and linearly “hot spots”–number–depended SERS properties. For a certain number of NPs, the number of “hot spots” and accordingly enhancement factor of Au NAs can be quantitatively evaluated, which open a new avenue for quantitative analysis based on SERS technique. PMID:26581251

The hidden life of integrative and conjugative elements

PubMed Central

Delavat, François; Miyazaki, Ryo; Carraro, Nicolas; Pradervand, Nicolas

2017-01-01

Abstract Integrative and conjugative elements (ICEs) are widespread mobile DNA that transmit both vertically, in a host-integrated state, and horizontally, through excision and transfer to new recipients. Different families of ICEs have been discovered with more or less restricted host ranges, which operate by similar mechanisms but differ in regulatory networks, evolutionary origin and the types of variable genes they contribute to the host. Based on reviewing recent experimental data, we propose a general model of ICE life style that explains the transition between vertical and horizontal transmission as a result of a bistable decision in the ICE–host partnership. In the large majority of cells, the ICE remains silent and integrated, but hidden at low to very low frequencies in the population specialized host cells appear in which the ICE starts its process of horizontal transmission. This bistable process leads to host cell differentiation, ICE excision and transfer, when suitable recipients are present. The ratio of ICE bistability (i.e. ratio of horizontal to vertical transmission) is the outcome of a balance between fitness costs imposed by the ICE horizontal transmission process on the host cell, and selection for ICE distribution (i.e. ICE ‘fitness’). From this emerges a picture of ICEs as elements that have adapted to a mostly confined life style within their host, but with a very effective and dynamic transfer from a subpopulation of dedicated cells. PMID:28369623

The importance of regulatory ubiquitination in cancer and metastasis

PubMed Central

Gallo, L. H.; Ko, J.; Donoghue, D. J.

2017-01-01

ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

DNA-encoded chemistry: enabling the deeper sampling of chemical space.

PubMed

Goodnow, Robert A; Dumelin, Christoph E; Keefe, Anthony D

2017-02-01

DNA-encoded chemical library technologies are increasingly being adopted in drug discovery for hit and lead generation. DNA-encoded chemistry enables the exploration of chemical spaces four to five orders of magnitude more deeply than is achievable by traditional high-throughput screening methods. Operation of this technology requires developing a range of capabilities including aqueous synthetic chemistry, building block acquisition, oligonucleotide conjugation, large-scale molecular biological transformations, selection methodologies, PCR, sequencing, sequence data analysis and the analysis of large chemistry spaces. This Review provides an overview of the development and applications of DNA-encoded chemistry, highlighting the challenges and future directions for the use of this technology.

Recombinant Clone Heterogeneity in ESCHERICHIA COLI Conjunction: Effect of Ph and Partially Replicated Recipient Deoxyribonucleic Acid

PubMed Central

Ou, Jonathan T.

1975-01-01

At pH 6.8, a substantial fraction of recombinant colonies obtained from conjugation with an HfrH donor contained multiple recombinant classes in a single colony (polygenotypic colony). In contrast, when the conjugation was performed at pH 7.6, the number of polygenotypic colonies was drastically reduced, and the recombinant colonies were predominantly monogenotypic or digenotypic. Genetic analysis revealed that the digenotypic recombinants differ in those donor markers near the origin of DNA replication but share those donor markers near the terminus. This integration pattern suggests that the formation of digenotypic recombinants involves recombination of a single copy of the exogenome with a partially replicated recipient DNA molecule. This suggestion was supported by examination of the genotype of recombinant colonies recovered from crosses with an HfrKL96 donor which was derived from HfrH but transfers its chromosome in the reverse direction. PMID:8360

Enhancing performing characteristics of organic semiconducting films by improved solution processing

DOEpatents

Bazan, Guillermo C; Moses, Daniel; Peet, Jeffrey; Heeger, Alan J

2014-05-13

Improved processing methods for enhanced properties of conjugated polymer films are disclosed, as well as the enhanced conjugated polymer films produced thereby. Addition of low molecular weight alkyl-containing molecules to solutions used to form conjugated polymer films leads to improved photoconductivity and improvements in other electronic properties. The enhanced conjugated polymer films can be used in a variety of electronic devices, such as solar cells and photodiodes.

RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway.

PubMed

Xie, Jenny; Kim, Hyungjin; Moreau, Lisa A; Puhalla, Shannon; Garber, Judy; Al Abo, Muthana; Takeda, Shunichi; D'Andrea, Alan D

2015-04-01

The Fanconi anemia/BRCA (FA/BRCA) pathway is a DNA repair pathway that is required for excision of DNA interstrand cross-links. The 17 known FA proteins, along with several FA-associated proteins (FAAPs), cooperate in this pathway to detect, unhook, and excise DNA cross-links and to subsequently repair the double-strand breaks generated in the process. In the current study, we identified a patient with FA with a point mutation in FANCA, which encodes a mutant FANCA protein (FANCAI939S). FANCAI939S failed to bind to the FAAP20 subunit of the FA core complex, leading to decreased stability. Loss of FAAP20 binding exposed a SUMOylation site on FANCA at amino acid residue K921, resulting in E2 SUMO-conjugating enzyme UBC9-mediated SUMOylation, RING finger protein 4-mediated (RNF4-mediated) polyubiquitination, and proteasome-mediated degradation of FANCA. Mutation of the SUMOylation site of FANCA rescued the expression of the mutant protein. Wild-type FANCA was also subject to SUMOylation, RNF4-mediated polyubiquitination, and degradation, suggesting that regulated release of FAAP20 from FANCA is a critical step in the normal FA pathway. Consistent with this model, cells lacking RNF4 exhibited interstrand cross-linker hypersensitivity, and the gene encoding RNF4 was epistatic with the other genes encoding members of the FA/BRCA pathway. Together, the results from our study underscore the importance of analyzing unique patient-derived mutations for dissecting complex DNA repair processes.

RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway

PubMed Central

Xie, Jenny; Kim, Hyungjin; Moreau, Lisa A.; Puhalla, Shannon; Garber, Judy; Al Abo, Muthana; Takeda, Shunichi; D’Andrea, Alan D.

2015-01-01

The Fanconi anemia/BRCA (FA/BRCA) pathway is a DNA repair pathway that is required for excision of DNA interstrand cross-links. The 17 known FA proteins, along with several FA-associated proteins (FAAPs), cooperate in this pathway to detect, unhook, and excise DNA cross-links and to subsequently repair the double-strand breaks generated in the process. In the current study, we identified a patient with FA with a point mutation in FANCA, which encodes a mutant FANCA protein (FANCAI939S). FANCAI939S failed to bind to the FAAP20 subunit of the FA core complex, leading to decreased stability. Loss of FAAP20 binding exposed a SUMOylation site on FANCA at amino acid residue K921, resulting in E2 SUMO-conjugating enzyme UBC9-mediated SUMOylation, RING finger protein 4–mediated (RNF4-mediated) polyubiquitination, and proteasome-mediated degradation of FANCA. Mutation of the SUMOylation site of FANCA rescued the expression of the mutant protein. Wild-type FANCA was also subject to SUMOylation, RNF4-mediated polyubiquitination, and degradation, suggesting that regulated release of FAAP20 from FANCA is a critical step in the normal FA pathway. Consistent with this model, cells lacking RNF4 exhibited interstrand cross-linker hypersensitivity, and the gene encoding RNF4 was epistatic with the other genes encoding members of the FA/BRCA pathway. Together, the results from our study underscore the importance of analyzing unique patient-derived mutations for dissecting complex DNA repair processes. PMID:25751062

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate.

PubMed

Puthenveetil, Sujiet; He, Haiyin; Loganzo, Frank; Musto, Sylvia; Teske, Jesse; Green, Michael; Tan, Xingzhi; Hosselet, Christine; Lucas, Judy; Tumey, L Nathan; Sapra, Puja; Subramanyam, Chakrapani; O'Donnell, Christopher J; Graziani, Edmund I

2017-01-01

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

NASA Astrophysics Data System (ADS)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-09-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

Empirical Modeling of Physiochemical Immune Response of Multilayer Zinc Oxide Nanomaterials under UV Exposure to Melanoma and Foreskin Fibroblasts

NASA Astrophysics Data System (ADS)

Fakhar-E-Alam, Muhammad; Akram, M. Waseem; Iqbal, Seemab; Alimgeer, K. S.; Atif, M.; Sultana, K.; Willander, M.; Wang, Zhiming M.

2017-04-01

Carcinogenesis is a complex molecular process starting with genetic and epigenetic alterations, mutation stimulation, and DNA modification, which leads to proteomic adaptation ending with an uncontrolled proliferation mechanism. The current research focused on the empirical modelling of the physiological response of human melanoma cells (FM55P) and human foreskin fibroblasts cells (AG01518) to the multilayer zinc oxide (ZnO) nanomaterials under UV-A exposure. To validate this experimental scheme, multilayer ZnO nanomaterials were grown on a femtotip silver capillary and conjugated with protoporphyrin IX (PpIX). Furthermore, PpIX-conjugated ZnO nanomaterials grown on the probe were inserted into human melanoma (FM55P) and foreskin fibroblasts cells (AG01518) under UV-A light exposure. Interestingly, significant cell necrosis was observed because of a loss in mitochondrial membrane potential just after insertion of the femtotip tool. Intense reactive oxygen species (ROS) fluorescence was observed after exposure to the ZnO NWs conjugated with PpIX femtotip model under UV exposure. Results were verified by applying several experimental techniques, e.g., ROS detection, MTT assay, and fluorescence spectroscopy. The present work reports experimental modelling of cell necrosis in normal human skin as well as a cancerous tissue. These obtained results pave the way for a more rational strategy for biomedical and clinical applications.

The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butz, Nicole; Ruetz, Stephan; Natt, Francois

2005-02-15

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found tomore » be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.« less

Design of molecular beacons as signaling probes for adenosine triphosphate detection in cancer cells based on chemiluminescence resonance energy transfer.

PubMed

Zhang, Shusheng; Yan, Yameng; Bi, Sai

2009-11-01

In the present study, binary and triplex DNA molecular beacons, as signaling probes based on a luminol-H(2)O(2)-horseradish peroxidase (HRP)-fluorescein chemiluminescence resonance energy transfer (CRET) system and structure-switching aptamers for highly sensitive detection of small molecules, are developed using adenosine triphosphate (ATP) as a model analyte to demonstrate the generality of the strategy. This CRET process occurs from donor luminol to acceptor fluorescein, which is oxidized by H(2)O(2) and catalyzed by HRP. DNA aptamer for ATP is first attached on the surface of magnetic nanoparticles (MNPs). The cDNA linker has an extension that hybridizes with two other DNAs (LumAuNP-DNA and F-DNA) or three other DNAs (HRP-DNA, LumAuNP-DNA, and F-DNA) to fabricate CRET-BMBP-MNP or CRET-TMBP-MNP conjugates that provide the CRET signals. Thus, in the absence of ATP, when the MNPs are removed from the solution, they also take with them the linker DNA and the CRET signal probes, and no CRET signal can be detected. However, when ATP is introduced, a competition for the ATP aptamer between ATP and the cDNA linker occurs. As a result, CRET-BMBP and CRET-TMBP are forced to dissociate from the MNP surface based on the structure switching of the aptamer. The CRET signals are proportional to the concentration of ATP. In order to accelerate the rate of the aptamer structure-switching process, an invader DNA is introduced into the proposed strategy. The present CRET system provides a low detection limit of 1.1 x 10(-7) and 3.2 x 10(-7) M for ATP detection by BMBP and TMBP, respectively, which also exhibits a good selectivity for ATP detection. Sample assays of ATP in K562 leukemia cells and 4T1 breast cancer cells confirm the reliability and practicality of the protocol, which reveal a good prospect of this platform for biological sample analysis.

Ligand-functionalized degradable polyplexes formed by cationic poly(aspartic acid)-grafted chitosan-cyclodextrin conjugates

NASA Astrophysics Data System (ADS)

Song, Hai-Qing; Li, Rui-Quan; Duan, Shun; Yu, Bingran; Zhao, Hong; Chen, Da-Fu; Xu, Fu-Jian

2015-03-01

Polypeptide-based degradable polyplexes attracted considerable attention in drug delivery systems. Polysaccharides including cyclodextrin (CD), dextran, and chitosan (CS) were readily grafted with cationic poly(aspartic acid)s (PAsps). To further enhance the transfection performances of PAsp-based polyplexes, herein, different types of ligand (folic acid, FA)-functionalized degradable polyplexes were proposed based on the PAsp-grafted chitosan-cyclodextrin conjugate (CCPE), where multiple β-CDs were tied on a CS chain. The FA-functionalized CCPE (i.e., CCPE-FA) was obtained via a host-guest interaction between the CD units of CCPE and the adamantane (Ad) species of Ad-modified FA (Ad-FA). The resulting CCPE/pDNA, CCPE-FA/pDNA, and ternary CCPE-FA/CCPE/pDNA (prepared by layer-by-layer assembly) polyplexes were investigated in detail using different cell lines. The CCPE-based polyplexes displayed much higher transfection efficiencies than the CS-based polyplexes reported earlier by us. The ternary polyplexes of CCPE-FA/CCPE/pDNA produced excellent gene transfection abilities in the folate receptor (FR)-positive tumor cells. This work would provide a promising means to produce highly efficient polyplexes for future gene therapy applications.Polypeptide-based degradable polyplexes attracted considerable attention in drug delivery systems. Polysaccharides including cyclodextrin (CD), dextran, and chitosan (CS) were readily grafted with cationic poly(aspartic acid)s (PAsps). To further enhance the transfection performances of PAsp-based polyplexes, herein, different types of ligand (folic acid, FA)-functionalized degradable polyplexes were proposed based on the PAsp-grafted chitosan-cyclodextrin conjugate (CCPE), where multiple β-CDs were tied on a CS chain. The FA-functionalized CCPE (i.e., CCPE-FA) was obtained via a host-guest interaction between the CD units of CCPE and the adamantane (Ad) species of Ad-modified FA (Ad-FA). The resulting CCPE/pDNA, CCPE-FA/pDNA, and ternary CCPE-FA/CCPE/pDNA (prepared by layer-by-layer assembly) polyplexes were investigated in detail using different cell lines. The CCPE-based polyplexes displayed much higher transfection efficiencies than the CS-based polyplexes reported earlier by us. The ternary polyplexes of CCPE-FA/CCPE/pDNA produced excellent gene transfection abilities in the folate receptor (FR)-positive tumor cells. This work would provide a promising means to produce highly efficient polyplexes for future gene therapy applications. Electronic supplementary information (ESI) available: 1H NMR assay, synthetic route of Ad-FA, AFM images and cellular internalization rate can be found in ESI. See DOI: 10.1039/c4nr07515c

From Bioengineering to CRISPR/Cas9 – A Personal Retrospective of 20 Years of Research in Programmable Genome Targeting

PubMed Central

Jeltsch, Albert

2018-01-01

Genome targeting of restriction enzymes and DNA methyltransferases has many important applications including genome and epigenome editing. 15–20 years ago, my group was involved in the development of approaches for programmable genome targeting, aiming to connect enzymes with an oligodeoxynucleotide (ODN), which could form a sequence-specific triple helix at the genomic target site. Importantly, the target site of such enzyme-ODN conjugate could be varied simply by altering the ODN sequence promising great applicative values. However, this approach was facing many problems including the preparation and purification of the enzyme-ODN conjugates, their efficient delivery into cells, slow kinetics of triple helix formation and the requirement of a poly-purine target site sequence. Hence, for several years genome and epigenome editing approaches mainly were based on Zinc fingers and TAL proteins as targeting devices. More recently, CRISPR/Cas systems were discovered, which use a bound RNA for genome targeting that forms an RNA/DNA duplex with one DNA strand of the target site. These systems combine all potential advantages of the once imagined enzyme-ODN conjugates and avoid all main disadvantageous. Consequently, the application of CRISPR/Cas in genome and epigenome editing has exploded in recent years. We can draw two important conclusions from this example of research history. First, evolution still is the better bioengineer than humans and, whenever tested in parallel, natural solutions outcompete engineered ones. Second, CRISPR/Cas system were discovered in pure, curiosity driven, basic research, highlighting that it is basic, bottom-up research paving the way for fundamental innovation. PMID:29434619

A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine

PubMed Central

Lee, HyungJae; Choi, Mihye; Hwang, Sang-Hyun; Cho, Youngnam

2018-01-01

Purpose: As human papillomavirus (HPV) is primarily responsible for the development of cervical cancer, significant efforts have been devoted to develop novel strategies for detecting and identifying HPV DNA in urine. The analysis of target DNA sequences in urine offers a potential alternative to conventional methods as a non-invasive clinical screening and diagnostic assessment tool for the detection of HPV. However, the lack of efficient approaches to isolate and directly detect HPV DNA in urine has restricted its potential clinical use. In this study, we demonstrated a novel approach of using polyethylenimine-conjugated magnetic polypyrrole nanowires (PEI-mPpy NWs) for the extraction, identification, and PCR-free colorimetric detection of high-risk strains of HPV DNA sequences, particularly HPV-16 and HPV-18, in urine specimens of cervical cancer patients. Materials and Methods: We fabricated and characterized polyethylenimine-conjugated magnetic nanowires (PEI/mPpy NWs). PEI/mPpy NWs-based HPV DNA isolation and detection strategy appears to be a cost-effective and practical technology with greater sensitivity and accuracy than other urine-based methods. Results: The analytical and clinical performance of PEI-mPpy NWs was evaluated and compared with those of cervical swabs, demonstrating a superior type-specific concordance rate of 100% between urine and cervical swabs, even when using a small volume of urine (300 µL). Conclusion: We envision that PEI-mPpy NWs provide substantive evidence for clinical diagnosis and management of HPV-associated disease with their excellent performance in the recovery and detection of HPV DNA from minimal amounts of urine samples. PMID:29290816

Synthesis of Sequence-Specific DNA-Protein Conjugates via a Reductive Amination Strategy

PubMed Central

Wickramaratne, Susith; Mukherjee, Shivam; Villalta, Peter W.; Schärer, Orlando D.; Tretyakova, Natalia

2013-01-01

DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to bis-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their super-bulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty of generating site-specific DNA substrates representative of DPC lesions formed in vivo. In the present study, a novel approach involving post-synthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides. PMID:23885807

Investigating bioconjugation by atomic force microscopy

PubMed Central

2013-01-01

Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures. PMID:23855448

Investigating bioconjugation by atomic force microscopy.

PubMed

Tessmer, Ingrid; Kaur, Parminder; Lin, Jiangguo; Wang, Hong

2013-07-15

Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures.

Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease.

PubMed

Nelson, Margaret-Ann M; Baba, Shahid P; Anderson, Ethan J

2017-04-01

Aldehydes are continuously formed in biological systems through enzyme-dependent and spontaneous oxidation of lipids, glucose, and primary amines. These highly reactive, biogenic electrophiles can become toxic via covalent modification of proteins, lipids and DNA. Thus, agents that scavenge aldehydes through conjugation have therapeutic value for a number of major cardiovascular diseases. Several commonly-prescribed drugs (e.g., hydralazine) have been shown to have potent aldehyde-conjugating properties which may contribute to their beneficial effects. Herein, we briefly describe the major sources and toxicities of biogenic aldehydes in cardiovascular system, and provide an overview of drugs that are known to have aldehyde-conjugating effects. Some compounds of phytochemical origin, and histidyl-dipeptides with emerging therapeutic value in this area are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytotoxicity Effects of Different Surfactant Molecules Conjugated to Carbon Nanotubes on Human Astrocytoma Cells

NASA Astrophysics Data System (ADS)

Dong, Lifeng; Witkowski, Colette M.; Craig, Michael M.; Greenwade, Molly M.; Joseph, Katherine L.

2009-12-01

Phase contrast and epifluorescence microscopy were utilized to monitor morphological changes in human astrocytoma cells during a time-course exposure to single-walled carbon nanotube (SWCNT) conjugates with different surfactants and to investigate sub-cellular distribution of the nanotube conjugates, respectively. Experimental results demonstrate that cytotoxicity of the nanotube/surfactant conjugates is related to the toxicity of surfactant molecules attached on the nanotube surfaces. Both sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) are toxic to cells. Exposure to CNT/SDS conjugates (0.5 mg/mL) for less than 5 min caused changes in cell morphology resulting in a distinctly spherical shape compared to untreated cells. In contrast, sodium cholate (SC) and CNT/SC did not affect cell morphology, proliferation, or growth. These data indicate that SC is an environmentally friendly surfactant for the purification and dispersion of SWCNTs. Epifluorescence microscopy analysis of CNT/DNA conjugates revealed distribution in the cytoplasm of cells and did not show adverse effects on cell morphology, proliferation, or viability during a 72-h incubation. These observations suggest that the SWCNTs could be used as non-viral vectors for diagnostic and therapeutic molecules across the blood-brain barrier to the brain and the central nervous system.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes.

PubMed

Ni, Lisheng; Jensen, Slade O; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M; Guan, Fiona H X; Brown, Melissa H; Skurray, Ronald A; Firth, Neville; Schumacher, Maria A

2009-11-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon-helix-helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

PubMed Central

Ni, Lisheng; Jensen, Slade O.; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M.; Guan, Fiona H. X.; Brown, Melissa H.; Skurray, Ronald A.; Firth, Neville; Schumacher, Maria A.

2009-01-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes. PMID:19759211

Characterization of human translesion DNA synthesis across a UV-induced DNA lesion

PubMed Central

Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

2016-01-01

Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand. DOI: http://dx.doi.org/10.7554/eLife.19788.001 PMID:27770570

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vega-Arroyo, M.; LeBreton, P. R.; Zapol, P.

Photoinduced charge separation in triads of DNA covalently linked to an anatase nanoparticle via a dopamine bridge was studied by ab initio calculations of the oxidation potentials of carboxyl-DNA trimers and the TiO2/dopamine complex. Conjugation of dopamine to the TiO2 surface results in a lower oxidation potential of the complex relative to the surface and in localization of photogenerated holes on dopamine, while photogenerated electrons are excited into the conduction band of TiO2. Linking dopamine to the DNA trimers at the 5? end of the oligonucleotide may lead to further hole migration to the DNA. Calculations show that for severalmore » different sequences hole migration is favorable in double stranded DNA and unfavorable in single-stranded DNA. This extended charge separation was shown to follow from the redox properties of DNA sequence rather than from the modification of DNA's electron donating properties by the dopamine linker, which explains experimental observations.« less

Complementation of a red-light-indifferent cyanobacterial mutant.

PubMed Central

Chiang, G G; Schaefer, M R; Grossman, A R

1992-01-01

Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation. Images PMID:1409650

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

PubMed Central

Saveliev, S V; Cox, M M

1994-01-01

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5). Images PMID:7838724

Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

PubMed

Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

2013-09-20

Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.

Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

PubMed

Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

2014-08-20

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

Development and Synthesis of DNA-Encoded Benzimidazole Library.

PubMed

Ding, Yun; Chai, Jing; Centrella, Paolo A; Gondo, Chenaimwoyo; DeLorey, Jennifer L; Clark, Matthew A

2018-04-25

Encoded library technology (ELT) is an effective approach to the discovery of novel small-molecule ligands for biological targets. A key factor for the success of the technology is the chemical diversity of the libraries. Here we report the development of DNA-conjugated benzimidazoles. Using 4-fluoro-3-nitrobenzoic acid as a key synthon, we synthesized a 320 million-member DNA-encoded benzimidazole library using Fmoc-protected amino acids, amines and aldehydes as diversity elements. Affinity selection of the library led to the discovery of a novel, potent and specific antagonist of the NK3 receptor.

Processing Conjugated-Diene-Containing Polymers

NASA Technical Reports Server (NTRS)

Bell, Vernon L.; Havens, Stephen J.

1987-01-01

Diels-Alder reaction used to cross-linked thermoplastics. Process uses Diels-Alder reaction to cross-link and/or extend conjugated-diene-containing polymers by reacting them with bis-unsaturated dienophiles results in improved polymer properties. Quantities of diene groups required for cross-linking varies from very low to very high concentrations. Process also used to extend, or build up molecular weights of, low-molecular-weight linear polymers with terminal conjugated dienic groups.

Role of isothiocyanate conjugate of pterostilbene on the inhibition of MCF-7 cell proliferation and tumor growth in Ehrlich ascitic cell induced tumor bearing mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nikhil, Kumar; Sharan, Shruti; Chakraborty, Ajanta

2014-01-15

Naturally occurring pterostilbene (PTER) and isothiocyanate (ITC) attract great attention due to their wide range of biological properties, including anti-cancer, anti-leukemic, anti-bacterial and anti-inflammatory activities. A novel class of hybrid compound synthesized by introducing an ITC moiety on PTER backbone was evaluated for its anti-cancer efficacy in hormone-dependent breast cancer cell line (MCF-7) in vitro and Ehrlich ascitic tumor bearing mice model in vivo. The novel hybrid molecule showed significant in vitro anti-cancer activity (IC{sub 50}=25±0.38) when compared to reference compound PTER (IC{sub 50}=65±0.42). The conjugate molecule induced both S and G2/M phase cell cycle arrest as indicated by flowmore » cytometry analysis. In addition, the conjugate induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-9, release of cytochrome-c into cytosol and increased Bax: Bcl-2 ratio. The conjugate also suppressed the phosphorylation of Akt and ERK. The conjugate induced cell death was significantly increased in presence of A6730 (a potent Akt1/2 kinase inhibitor) and PD98059 (a specific ERK inhibitor). Moreover, the conjugated PTER inhibited tumor growth in Ehrlich ascitic cell induced tumor bearing mice as observed by reduction in tumor volume compared to untreated animals. Collectively, the pro-apoptotic effect of conjugate is mediated through the activation of caspases, and is correlated with the blockade of the Akt and ERK signaling pathways in MCF-7 cells. - Highlights: • Conjugate was prepared by appending isothiocyanate moiety on pterostilbene backbone. • Conjugate showed anticancer effects at comparatively lower dose than pterostilbene. • Conjugate caused blockage of the Akt and ERK signaling pathways in MCF-7 cells. • Conjugate significantly reduced solid tumor volume as compared to pterostilbene.« less

Streptococcus gordonii pheromone s.g.cAM373 may influence the reservoir of antibiotic resistance determinants of Enterococcus faecalis origin in the oral metagenome.

PubMed

Mansfield, Jillian M; Herrmann, Paul; Jesionowski, Amy M; Vickerman, M Margaret

2017-11-01

Streptococcus gordonii produces a pheromone heptapeptide, s.g.cAM373, which induces a conjugative mating response in Enterococcus faecalis cells carrying the responsive plasmid, pAM373. We investigated the extent of this intergeneric signaling on DNA acquisition by streptococcal species likely to cohabit oral biofilms. E. faecalis/pAM373/pAMS470 cells were incubated with synthetic s.g.cAM373, reverse peptide s.g.cAM373-R, or peptide-free medium and examined for their abilities to transfer plasmid DNA to streptococcal species in the presence of DNase. Preinduction of E. faecalis donors with s.g.cAM373 resulted in transconjugation frequencies in non-pheromone producing strains of Streptococcus mutans, Streptococcus sanguinis, Streptococcus anginosus, and Streptococcus suis that were significantly higher than frequencies when donors were preincubated with s.g.cAM373-R or medium alone. Peptide-mediated communication between commensal streptococci and E. faecalis carrying pheromone-responsive plasmids may facilitate conjugative DNA transfer to bystander species, and influence the reservoir of antibiotic resistance determinants of enterococcal origin in the oral metagenome.

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins.

PubMed

Wang, Feng; Zhang, Xiaohan; Liu, Yibo; Lin, Zhi Yuan William; Liu, Biwu; Liu, Juewen

2016-09-19

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo-TEM, and ζ-potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3 O4 , TiO2 , ZrO2 , Y2 O3 , ITO, In2 O3 , and Mn2 O3 ) form supported bilayers only with DOCP. Type 2 (SiO2 ) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3 O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides.

PubMed

Fan, P W; Zhang, F; Bolton, J L

2000-01-01

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half-life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction mechanism likely involves an episulfonium ion intermediate which may contribute to the potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen, 4-hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about 3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7 cells. Finally, these data suggest that quinone methide formation may not make a significant contribution to the cytotoxic and genotoxic effects of tamoxifen and toremifene.

DNA-polymer micelles as nanoparticles with recognition ability.

PubMed

Talom, Renée Mayap; Fuks, Gad; Kaps, Leonard; Oberdisse, Julian; Cerclier, Christel; Gaillard, Cédric; Mingotaud, Christophe; Gauffre, Fabienne

2011-11-25

The Watson-Crick binding of DNA single strands is a powerful tool for the assembly of nanostructures. Our objective is to develop polymer nanoparticles equipped with DNA strands for surface-patterning applications, taking advantage of the DNA technology, in particular, recognition and reversibility. A hybrid DNA copolymer is synthesized through the conjugation of a ssDNA (22-mer) with a poly(ethylene oxide)-poly(caprolactone) diblock copolymer (PEO-b-PCl). It is shown that, in water, the PEO-b-PCl-ssDNA(22) polymer forms micelles with a PCl hydrophobic core and a hydrophilic corona made of PEO and DNA. The micelles are thoroughly characterized using electron microscopy (TEM and cryoTEM) and small-angle neutron scattering. The binding of these DNA micelles to a surface through DNA recognition is monitored using a quartz crystal microbalance and imaged by atomic force microscopy. The micelles can be released from the surface by a competitive displacement event. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

PubMed

Gubu, Amu; Li, Long; Ning, Yan; Zhang, Xiaoyun; Lee, Seonghyun; Feng, Mengke; Li, Qiang; Lei, Xiaoguang; Jo, Kyubong; Tang, Xinjing

2018-04-17

Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting MED1 LxxLL Motifs for Tissue-Selective Treatment of Human Breast Cancer

DTIC Science & Technology

2013-09-01

colleagues have successfully conjugated malachite green aptamer to RNA nanoparticles characterized by a 3WJ pRNA motif. The in vitro experiment indi- cated...DNA/RNA sequence FIGURE 19.5 Diagram of RNA nanoparticle harboring malachite green aptamer, survivin siRNA and folate-DNA/RNA sequence for targeting...of RNA Aptamer to RNA Nanoparticles (Figure 19.5; Shu et al. 2011). The sequence for the malachite green aptamer nanoparticle was rationally designed

[Non-viral gene therapy approach for regenerative recovery of skin wounds in mammals].

PubMed

Efremov, A M; Dukhovlinov, I V; Dizhe, E B; Burov, S V; Leko, M V; Akif'ev, B N; Mogilenko, D A; Ivanov, I A; Perevozchikov, A P; Orlov, S V

2010-01-01

The rate and character of skin tissue regeneration after wounds, burns and other traumas depend on the cell proliferation within damaged area. Acceleration of healing by stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment consisting in the transfer of genes encoding mitogenic growth factors to wound area. The most important step in the development of gene therapy approaches is the design of gene delivery tools. In spite of high efficacy of viral vectors, the non-viral means have some preferences (low toxicity, low immunogenity, safety and the absence of backside effects). Among non-viral gene delivery tools, molecular conjugates are the most popular because of their efficacy, simplicity, and the capacity to the targeted gene transfer. In the present work we have developed two molecular conjugates--NLS-TSF7 and NLS-TSF12 consisting of the modified signal of nuclear localization of T-antigen of SV40 virus (cationic part) and the peptide ligands of mammalian transferrin receptor (ligand part). These conjugates bind to plasmid DNA with formation of polyelectrolytic complexes and are capable to deliver plasmid DNA into cells expressing transferrin receptors by receptor-mediated endocytosis. Transfer of the expression vector of luciferase gene in the complex with molecular conjugate NLS-TSF7 to murine surface tissues led to about 100 fold increasing of luciferase activity in comparison with the transfer of free expression vector. Treatment of slash wounds in mice with the complexes of expression vector of synthetic human gene encoding insulin-like growth factor 1 with molecular conjugates NLS-TSF7 led to acceleration of healing in comparison with mice treated with free expression vector. The results obtained confirm the high efficiency of the developed regenerative gene therapy approach for the treatment of damaged skin tissues in mammals.

Cationic DOPC-Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery.

PubMed

Pierrat, Philippe; Casset, Anne; Didier, Pascal; Kereselidze, Dimitri; Lux, Marie; Pons, Françoise; Lebeau, Luc

2016-09-15

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC-C12 E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12 E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12 E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic-lipid-mediated gene transfer in animals. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exciton transport in π-conjugated polymers with conjugation defects.

PubMed

Meng, Ruixuan; Li, Yuan; Li, Chong; Gao, Kun; Yin, Sun; Wang, Luxia

2017-09-20

In π-conjugated polymers for photovoltaic applications, intrinsic conjugation defects are known to play crucial roles in impacting exciton transport after photoexcitation. However, the understanding of the associated microscopic processes still remains limited. Here, we present a theoretical investigation of the effects of different conjugation defects on the dynamics of exciton transport in two linearly coupled poly(p-phenylene vinylene) (PPV) molecules. The model system is constructed by employing an extended version of the Su-Schrieffer-Heeger model and the exciton behaviors are simulated by means of a quantum nonadiabatic dynamics. We identify two types of conjugation defects, i.e., weakening conjugation and strengthening conjugation, which are demonstrated to play different roles in impacting the dynamics of exciton transport in the system. The weakening conjugation acts as an energy well inclined to trap a moving exciton, while the strengthening conjugation acts as an energy barrier inclined to block the exciton. We also systematically simulate both intrachain and interchain dynamics of exciton transport, and find that an exciton could experience a "short-time delaying", "trapping", "blocking", or "hopping" process, which is determined by the defect type, strength, and position. These findings provide a microscopic understanding of how the exciton transport dynamics can be impacted by conjugation defects in an actual polymer system.

Modulation of carcinogen bioavailability by immunisation with benzo[a]pyrene-conjugate vaccines.

PubMed

Grova, Nathalie; Prodhomme, Emmanuel J F; Schellenberger, Mario T; Farinelle, Sophie; Muller, Claude P

2009-06-24

Benzo[a]pyrene (B[a]P) conjugate vaccines based on ovalbumin, tetanus toxoid and diphtheria toxoid (DT) as carrier proteins were developed to investigate the effect of specific antibodies on the bioavailability of this ubiquitous carcinogen and its metabolites. After metabolic activation of this prototype carcinogen, B[a]P forms DNA adducts which initiate chemical carcinogenesis. B[a]P-DT conjugate induced the most robust immune response. The antibodies reacted not only with B[a]P but also with the proximate carcinogen 7,8-diol-B[a]P. Antibodies modulated the bioavailability of B[a]P and its metabolic activation in a dose-dependent manner by sequestration in the blood. Our results showed that this immune prophylactic strategy influences the pharmacokinetic of B[a]P and further studies to investigate their effects on chemical carcinogenesis are warranted.

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site.

PubMed

Abe, Yukiko; Nakagawa, Osamu; Yamaguchi, Rie; Sasaki, Shigeki

2012-06-01

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

PubMed

Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

2016-03-15

Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of an ultrasensitive PCR assay for polycyclic musk determination in fish.

PubMed

Zhang, Xiaohan; Zhuang, Huisheng

2018-05-01

Polycyclic musks (PCMs) in the aquatic environment and organisms have become an emerging environmental issue because of their potential risk. The most used method for polycyclic musk determination is gas chromatography-mass spectrometry (GC-MS) with different sample extractions, which are somewhat expensive to operate, complex and laborious. In this study, a novel and ultrasensitive real-time polymerase chain reaction (PCR) assay with multiple signal amplification of carboxylic-DNA by gold nanoparticle-polyamidoamine conjugation (Au-PAMAM) was developed for determining polycyclic musks in fish. Hapten and immunogen were specially prepared. Polyclonal antibodies were produced based on the optimal immunisation, and the antibodies were characterised. Due to PAMAM's unique nanostructure of numerous functional amino groups, polyclonal antibody and carboxylic-DNA were immobilised by Au-PAMAM conjugation to develop the antibody-Au-PAMAM-DNA probes, which were used as a signal DNA amplifier in the PCR system. Compared with real-time immuno-PCR, this biological probe-amplified immuno-PCR (BPAI-PCR) assay had higher sensitivity due to the probes' higher ratio of signal DNA. Finally, the BPAI-PCR assay was applied to analyse AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene,Tonalide) concentrations in fish samples in the range from 1 pg/L to 10 ng/L, giving an of LOD 0.61 pg/L. In general, due to the specificity of the antibody and novel nanoprobe design, this BPAI-PCR assay provided a potential way for trace analysis of AHTN in the aquatic organisms. The high concentrations of AHTN found in cultivated fish should encourage further toxicological studies.

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

2017-03-01

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

Preclinical evaluation of a Haemophilus influenzae type b conjugate vaccine process intended for technology transfer.

PubMed

Hamidi, Ahd; Verdijk, Pauline; Kreeftenberg, Hans

2014-01-01

Introduction of Haemophilus influenzae type b (Hib) vaccine in low- and middle-income countries has been limited by cost and availability of Hib conjugate vaccines for a long time. It was previously recognized by the Institute for Translational Vaccinology (Intravacc, originating from the former Vaccinology Unit of the National Institute of Public Health [RIVM] and the Netherlands Vaccine Institute [NVI]) that local production of a Hib conjugate vaccine would increase the affordability and sustainability of the vaccine and thereby help to speed up Hib introduction in these countries. A new affordable and a non-infringing production process for a Hib conjugate vaccine was developed, including relevant quality control tests, and the technology was transferred to a number of vaccine manufacturers in India, Indonesia, and China. As part of the Hib technology transfer project managed by Intravacc, a preclinical toxicity study was conducted in the Netherlands to test the safety and immunogenicity of this new Hib conjugate vaccine. The data generated by this study were used by the technology transfer partners to accelerate the clinical development of the new Hib conjugate vaccine. A repeated dose toxicity and local tolerance study in rats was performed to assess the reactogenicity and immunogenicity of a new Hib conjugate vaccine compared to a licensed vaccine. The results showed that the vaccine was well tolerated and immunogenic in rats, no major differences in both safety and immunogenicity in rats were found between the vaccine produced according to the production process developed by Intravacc and the licensed one. Rats may be useful to verify the immunogenicity of Hib conjugate vaccines and for preclinical evaluation. In general, nonclinical evaluation of the new Hib conjugate vaccine, including this proof of concept (safety and immunogenicity study in rats), made it possible for technology transfer partners, having implemented the original process with no changes in the manufacturing process and vaccine formulation, to start directly with phase 1 clinical trials.

Repercussions of imprisonment for conjugal violence: discourses of men 1

PubMed Central

de Sousa, Anderson Reis; Pereira, Álvaro; Paixão, Gilvânia Patrícia do Nascimento; Pereira, Nadirlene Gomes; Campos, Luana Moura; Couto, Telmara Menezes

2016-01-01

ABSTRACT Objective: to know the consequences that men experience related to incarceration by conjugal violence. Methods: qualitative study on 20 men in jail and indicted in criminal processes related to conjugal violence in a Court specialized in Family and Domestic Violence against women. The interviews were classified based on Collective Subject Discourse method, using NVIVO(r) software. Results: the collective discourse shows that the experience of preventive imprisonment starts a process of family dismantling, social stigma, financial hardship and psycho-emotional symptoms such as phobia, depression, hypertension, and headaches. Conclusion: due to the physical, mental and social consequences of the conjugal violence-related imprisonment experience, it is urgent to look carefully into the somatization process as well as to the prevention strategies regarding this process. PMID:27982312

In Vitro Targeted Photodynamic Therapy with a Pyropheophorbide-a Conjugated Inhibitor of Prostate Specific Membrane Antigen

PubMed Central

Liu, Tiancheng; Wu, Lisa Y.; Choi, Joseph K.; Berkman, Clifford E.

2009-01-01

BACKROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on photosensitizer-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 h by HOE33342/PI double-staining, becoming more intense by 4 h. Evidence for the apoptotic caspase cascade being activated was based on the appearance of PARP p85 fragment. TUNEL assay detected DNA fragmentation 16 h post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double-staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. PMID:19142895

In vitro targeted photodynamic therapy with a pyropheophorbide--a conjugated inhibitor of prostate-specific membrane antigen.

PubMed

Liu, Tiancheng; Wu, Lisa Y; Choi, Joseph K; Berkman, Clifford E

2009-05-01

The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. (c) 2009 Wiley-Liss, Inc.

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay.

PubMed

Liu, Daojun; Perdue, Robbyn K; Sun, Li; Crooks, Richard M

2004-07-06

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.

A nanophosphor-based method for selective DNA recovery in Synthosomes.

PubMed

Nallani, Madhavan; Onaca, Ozana; Gera, Nimish; Hildenbrand, Karlheinz; Hoheisel, Werner; Schwaneberg, Ulrich

2006-01-01

A nanocompartment system composed of an ABA triblock copolymer, where A is poly(dimethylsiloxane) and B is poly(2-methyloxazoline), has been developed for selective recovery and detection of DNA. Translocation of TAMRA-labeled complementary primers into the nanocompartment system has been achieved through two deletion mutants (FhuA Delta1-129; FhuA Delta1-160) of the channel protein FhuA. Translocation was monitored by fluorescence resonance energy transfer through hybridization of the TAMRA-labeled primer to the complementary sequence of a nanophosphor-DNA-conjugate, which reduces its half-life (FhuA Delta1-129, 16.0% reduced; FhuA Delta1-160, 39.0% reduced).

Chemical Biology Probes from Advanced DNA-encoded Libraries.

PubMed

Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

2016-02-19

The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs.

Binding of transcription termination protein nun to nascent RNA and template DNA.

PubMed

Watnick, R S; Gottesman, M E

1999-12-17

The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

Final Report: DOE Award Number: DE-SC0006398, University of CA, San Diego

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cha, Jennifer

The focus of the proposed research is to direct the assembly of single or binary nanoparticles into meso- or macroscale three-dimensional crystals of any desired configuration and crystallographic orientation without using prohibitively expensive lithographic processes. The epitaxial nucleation of defect-free, surface-bound bulk single crystals will revolutionize technologies for energy to generate new types of solar cells that yield maximum conversion efficiencies. It has been proposed that having a nanostructured bulk hetero-interface will enable efficient charge-carrier separations, similar to organic based heterojunction cells but with potential improvements, including thermal and long-term stability, tunability of energy levels, large adsorption coefficients and carriermore » multiplication. However, engineering such devices requires nanoscale control and ordering in both 2- and 3-dimensions over macroscopic areas and this has yet to be achieved. In Nature, bulk organic and inorganic materials are arranged into precise and ordered programmed assemblies through the sequestration of raw materials into confined spaces and association through highly specific non-covalent interactions between biomolecules. Using similar strategies, the proposed research will focus on confining metal and semiconductor nanocrystals to pre-determined surface patterns and controlling their arrangement through tunable, orthogonal biomolecular binding. Once a perfect two-dimensional seed layer has been constructed, successive layers of single nanocrystals will be nucleated epitaxially with long-range order and tunable crystallographic orientations. The proposed research exploits the ability of biomolecules to bind specific targets in a tunable, orthogonal, multivalent, and reversible manner to the arrangements of DNA-nanoparticle conjugates on chemically defined surfaces. Through careful balance of the attractive and repulsive forces between the particles, the array, and the outside surface, it is envisioned that single or mixed nanoparticles can be packed to adopt uniform crystal orientation in two and three dimensions from simple mixing and annealing of biomolecule-nanoparticle conjugates with biomolecule-stamped surfaces. To control the crystallographic alignment of each particle with its neighbors, the nanoparticles will be assembled using a mixture of non-covalent biomolecular interactions. To create solar cells in which layers of donor and acceptor nanocrystals that are not only oriented normal to the top and bottom electrodes but are also arranged in a checkerboard pattern, multicomponent nanocrystals (e.g. CdSe, CdTe) will be conjugated with biochemical linkers such that only interactions between the CdTe and CdSe promote particle packing within the array. The proposed research will: (1) elucidate the role of single and binary cooperative particle-DNA interactions in influencing nanoparticle crystallographic orientation in two and three dimensions; (2) understand how confinement of nanoparticles on patterned arrays of biomolecules and modification of the surrounding substrate can nucleate long-range order over macroscopic areas via predefined grain boundaries; and (3) synthesize and characterize DNA conjugated semiconductor nanocrystals and assemble them into 2- and 3-D binary superlattice arrays for photovoltaics.« less

Pulmonary delivery of respiratory syncytial virus DNA vaccines using macroaggregated albumin particles.

PubMed

Harcourt, Jennifer L; Anderson, Larry J; Sullender, Wayne; Tripp, Ralph A

2004-06-02

At present there is no safe and effective vaccine for respiratory syncytial virus (RSV). DNA vaccines encoding RSV surface glycoproteins are one option being examined. Current methods to deliver DNA vaccines generally require repeated high dose intramuscular or intradermal administration for effectiveness. In this study, we examine the efficacy of pulmonary DNA vaccination using low dose DNA vaccines encoding the RSV F glycoprotein conjugated to macroaggregated albumin (MAA-F). Single vaccination of BALB/c mice with 1 microg MAA-F was ineffective, however mice boosted with an additional 1 microg MAA-F, or vaccinated a single time with 10 microg MAA-F, developed substantially improved immunity associated with reduced viral titers, increased anti-F antibody responses, and enhanced Th1 and Th2 intracellular cytokine responses. This study shows that MAA may be a useful carrier for RSV DNA vaccines.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

The Reach of Linear Protein-DNA Dimerizers

PubMed Central

Stafford, Ryan L.; Dervan, Peter B.

2008-01-01

A protein-DNA dimerizer constructed from a DNA-binding pyrrole-imidazole polyamide and the peptide FYPWMK facilitates binding of the natural transcription factor Exd to an adjacent DNA site. Previous dimerizers have been constructed with the peptide attached to an internal pyrrole monomer in an overall branched oligomer. Linear oligomers constructed by attaching the peptide to the polyamide C-terminus expand the range of protein-DNA dimerization to six additional DNA sites. Replacing the FYPWMK hexapeptide with a WM dipeptide, which was previously functional in branched compounds, does not lead to a functional linear dimerizer. Instead, inserting an additional lysine generates a minimal, linear WMK tripeptide conjugate that maintains the activity of the larger FYPWMK dimerizers in a single DNA-binding site orientation. These studies provide insight into the importance of linker length and composition, binding site spacing and orientation, and the protein-binding domain content that are important for the optimization of protein DNA-dimerizers suitable for biological experiments. PMID:17949089

Magnetically responsive nanoparticles for drug delivery applications using low magnetic field strengths.

PubMed

McGill, Shayna L; Cuylear, Carla L; Adolphi, Natalie L; Osiński, Marek; Smyth, Hugh D C

2009-03-01

The purpose of this study is to investigate the potential of magnetic nanoparticles for enhancing drug delivery using a low oscillating magnetic field (OMF) strength. We investigated the ability of magnetic nanoparticles to cause disruption of a viscous biopolymer barrier to drug delivery and the potential to induce triggered release of drug conjugated to the surfaces of these particles. Various magnetic nanoparticles were screened for thermal response under a 295-kHz OMF with an amplitude of 3.1 kA/m. Based on thermal activity of particles screened, we selected the nanoparticles that displayed desired characteristics for evaluation in a simplified model of an extracellular barrier to drug delivery, using lambda DNA/HindIII. Results indicate that nanoparticles could be used to induce DNA breakage to enhance local diffusion of drugs, despite low temperatures of heating. Additional studies showed increased diffusion of quantum dots in this model by single-particle tracking methods. Bimane was conjugated to the surface of magnetic nanoparticles. Fluorescence and transmission electron microscope images of the conjugated nanoparticles indicated little change in the overall appearance of the nanoparticles. A release study showed greater drug release using OMF, while maintaining low bulk heating of the samples (T = 30 degrees C). This study indicates that lower magnetic field strengths may be successfully utilized for drug delivery applications as a method for drug delivery transport enhancement and drug release switches.

oriTfinder: a web-based tool for the identification of origin of transfers in DNA sequences of bacterial mobile genetic elements.

PubMed

Li, Xiaobin; Xie, Yingzhou; Liu, Meng; Tai, Cui; Sun, Jingyong; Deng, Zixin; Ou, Hong-Yu

2018-05-04

oriTfinder is a web server that facilitates the rapid identification of the origin of transfer site (oriT) of a conjugative plasmid or chromosome-borne integrative and conjugative element. The utilized back-end database oriTDB was built upon more than one thousand known oriT regions of bacterial mobile genetic elements (MGEs) as well as the known MGE-encoding relaxases and type IV coupling proteins (T4CP). With a combination of similarity searches for the oriTDB-archived oriT nucleotide sequences and the co-localization of the flanking relaxase homologous genes, the oriTfinder can predict the oriT region with high accuracy in the DNA sequence of a bacterial plasmid or chromosome in minutes. The server also detects the other transfer-related modules, including the potential relaxase gene, T4CP gene and the type IV secretion system gene cluster, and the putative genes coding for virulence factors and acquired antibiotic resistance determinants. oriTfinder may contribute to meeting the increasing demands of re-annotations for bacterial conjugative, mobilizable or non-transferable elements and aid in the rapid risk accession of disease-relevant trait dissemination in pathogenic bacteria of interest. oriTfinder is freely available to all users without any login requirement at http://bioinfo-mml.sjtu.edu.cn/oriTfinder.

Evaluation of functionalized SBA-15 to reduce patulin levels in apple juice

USDA-ARS?s Scientific Manuscript database

Patulin is a low molecular weight mycotoxin associated with the apple rotting fungus, Penicillium expansum. This toxin possesses an electrophilic conjugated double bond system susceptible to formation of harmful adducts with important biomolecules, such as glutathione, proteins, and DNA. In this stu...

In vivo metabolism and genotoxic effects of nitrated polycyclic aromatic hydrocarbons.

PubMed

Möller, L

1994-10-01

During incomplete combustion of organic matter, nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), are formed in a reaction that is catalyzed by a low pH. 2-Nitrofluorene (NF), a marker for nitro-PAHs, is metabolized in vivo by two different routes. After inhalation, potent mutagenic metabolites, hydroxylated nitrofluorenes (OH-NFs), are formed. The metabolites are distributed by systemic circulation. After oral administration, NF is reduced to the corresponding amine, a reaction mediated by the intestinal microflora. This metabolite is acetylated to 2-acetylaminofluorene (AAF), a potent carcinogen. Further ring-hydroxylation of AAF leads to detoxification and excretion. Induction of cytochrome P450s affects the metabolism, and more OH-NFs are formed. As a consequence, more mutagenic metabolites are found in the circulation. OH-NFs are excreted in the bile as, in terms of mutagenicity, totally harmless glucuronide conjugates. When these conjugates are excreted via the bile, intestinal beta-glucuronidase can liberate direct-acting mutagens in the intestine. Thus, inhalation of NF can lead to formation of potent mutagens in the intestine. NF is a direct-acting mutagen in bacterial assays and an initiator and promoter of the carcinogenic process, and gives rise to DNA adduct formation in laboratory animals.

Dendrimers as Nanocarriers for Nucleic Acid and Drug Delivery in Cancer Therapy

PubMed Central

Mendes, Livia Palmerston; Pan, Jiayi; Torchilin, Vladimir P.

2017-01-01

Dendrimers are highly branched polymers with easily modifiable surfaces. This makes them promising structures for functionalization and also for conjugation with drugs and DNA/RNA. Their architecture, which can be controlled by different synthesis processes, allows the control of characteristics such as shape, size, charge, and solubility. Dendrimers have the ability to increase the solubility and bioavailability of hydrophobic drugs. The drugs can be entrapped in the intramolecular cavity of the dendrimers or conjugated to their functional groups at their surface. Nucleic acids usually form complexes with the positively charged surface of most cationic dendrimers and this approach has been extensively employed. The presence of functional groups in the dendrimer’s exterior also permits the addition of other moieties that can actively target certain diseases and improve delivery, for instance, with folate and antibodies, now widely used as tumor targeting strategies. Dendrimers have been investigated extensively in the medical field, and cancer treatment is one of the greatest areas where they have been most used. This review will consider the main types of dendrimer currently being explored and how they can be utilized as drug and gene carriers and functionalized to improve the delivery of cancer therapy. PMID:28832535

Live-Cell Imaging of DNA Methylation Based on Synthetic-Molecule/Protein Hybrid Probe.

PubMed

Kumar, Naresh; Hori, Yuichiro; Kikuchi, Kazuya

2018-06-04

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

PubMed Central

Laoong-u-thai, Yanisa; Zhao, Baoping; Phongdara, Amornrat; Ako, Harry; Yang, Jinzeng

2009-01-01

Small ubiquitin-like modifiers (SUMO) work in a similar way as ubiquitin to alter the biological properties of a target protein by conjugation. A shrimp SUMO cDNA named LvSUMO-1 was identified in Litopenaeus vannamei. LvSUMO-1 cDNA contains a coding sequence of 282 nucleotides with untranslated regions of 37 bp at 5'-end and 347 bp at 3'-end, respectively. The deduced 93 amino acids exhibit 83% identity with the Western Honeybee SUMO-1, and more than 65% homologies with human and mouse SUMO-1. LvSUMO-1 mRNA is expressed in most L. vannamei tissues with the highest level in hepatopancrease. The mRNA expression of LvSUMO-1 over development stages in L. Vammamei is distinguished by a low level in nauplius stage and relatively high level in postlarva stage with continuous expression until juvenile stage. The LvSUMO-1 protein and its conjugated proteins are detected in both cytoplasm and nucleus in several tissues. Interestingly, LvSUMO-1 mRNA levels are high in abdominal muscle during the premolt stage, wherein it has significant activities of protein degradation, suggesting its possible role in the regulation of shrimp muscle protein degradation. PMID:19240809

Anthracycline-Formaldehyde Conjugates and Their Targeted Prodrugs

NASA Astrophysics Data System (ADS)

Koch, Tad H.; Barthel, Benjamin L.; Kalet, Brian T.; Rudnicki, Daniel L.; Post, Glen C.; Burkhart, David J.

The sequence of research leading to a proposal for anthracycline cross-linking of DNA is presented. The clinical anthracycline antitumor drugs are anthraquinones, and as such are redox active. Their redox chemistry leads to induction of oxidative stress and drug metabolites. An intermediate in reductive glycosidic cleavage is a quinone methide, once proposed as an alkylating agent of DNA. Subsequent research now implicates formaldehyde as a mediator of anthracycline-DNA cross-linking. The cross-link at 5'-GC-3' sites consists of a covalent linkage from the amino group of the anthracycline to the 2-amino group of the G-base through a methylene from formaldehyde, hydrogen bonding from the 9-OH to the G-base on the opposing strand, and hydrophobic interactions through intercalation of the anthraquinone. The combination of these interactions has been described as a virtual cross-link of DNA. The origin of the formaldehyde in vivo remains a mystery. In vitro, doxorubicin reacts with formaldehyde to give firstly a monomeric oxazolidine, doxazolidine, and secondly a dimeric oxazolidine, doxoform. Doxorubicin reacts with formaldehyde in the presence of salicylamide to give the N-Mannich base conjugate, doxsaliform. Doxsaliform is several fold more active in tumor cell growth inhibition than doxorubicin, but doxazolidine and doxoform are orders of magnitude more active than doxorubicin. Exploratory research on the potential for doxsaliform and doxazolidine as targeted cytotoxins is presented. A promising lead design is pentyl PABC-Doxaz, targeted to a carboxylesterase enzyme overexpressed in liver cancer cells and/or colon cancer cells.

Highly Efficient Flexible Hybrid Photovoltaic Cells Based on Low-Band-Gap Conjugated Polymers Sensitized by Nanoparticle-Grafted Carbon

DTIC Science & Technology

2010-09-01

modeling, synthesis , and characterization of several series functional and processable electro-active conjugated polymers with evolving frontier...tasks as a basic obligation of this award: Task #1. Low Band Gap Polymers The awardee (Professor Sun’s group at NSU) shall design, synthesis , and...design, modeling, synthesis , and characterizations of several series functional and processable electro-active conjugated polymers with evolving

Use of immuno assays during the development of a Hemophilus influenzae type b vaccine for technology transfer to emerging vaccine manufacturers.

PubMed

Hamidi, Ahd; Kreeftenberg, Hans

2014-01-01

Quality control of Hemophilus Influenzae type b (Hib) conjugate vaccines is mainly dependent on physicochemical methods. Overcoming sample matrix interference when using physicochemical tests is very challenging, these tests are therefore only used to test purified samples of polysaccharide, protein, bulk conjugate, and final product. For successful development of a Hib conjugate vaccine, several ELISA (enzyme-linked immunosorbent assay) methods were needed as an additional tool to enable testing of in process (IP) samples. In this paper, three of the ELISA's that have been very valuable during the process development, implementation and scaling up are highlighted. The PRP-ELISA, was a very efficient tool in testing in process (IP) samples generated during the development of the cultivation and purification process of the Hib-polysaccharide. The antigenicity ELISA, was used to confirm the covalent linkage of PRP and TTd in the conjugate. The anti-PRP IgG ELISA was developed as part of the immunogenicity test, used to demonstrate the ability of the Hib conjugate vaccine to elicit a T-cell dependent immune response in mice. ELISA methods are relatively cheap and easy to implement and therefore very useful during the development of polysaccharide conjugate vaccines.

Improving Analytical Characterization of Glycoconjugate Vaccines through Combined High-Resolution MS and NMR: Application to Neisseria meningitidis Serogroup B Oligosaccharide-Peptide Glycoconjugates.

PubMed

Yu, Huifeng; An, Yanming; Battistel, Marcos D; Cipollo, John F; Freedberg, Darón I

2018-04-17

Conjugate vaccines are highly heterogeneous in terms of glycosylation sites and linked oligosaccharide length. Therefore, the characterization of conjugate vaccines' glycosylation state is challenging. However, improved product characterization can lead to enhancements in product control and product quality. Here, we present a synergistic combination of high-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) for the analysis of glycoconjugates. We use the power of this strategy to characterize model polysaccharide conjugates and to demonstrate a detailed level of glycoproteomic analysis. These are first steps on model compounds that will help untangle the details of complex product characterization in conjugate vaccines. Ultimately, this strategy can be applied to enhance the characterization of polysaccharide conjugate vaccines. In this study, we lay the groundwork for the analysis of conjugate vaccines. To begin this effort, oligosaccharide-peptide conjugates were synthesized by periodate oxidation of an oligosaccharide of a defined length, α,2-8 sialic acid trimer, followed by a reductive amination, and linking the trimer to an immunogenic peptide from tetanus toxoid. Combined mass spectrometry and nuclear magnetic resonance were used to monitor each reaction and conjugation products. Complete NMR peak assignment and detailed MS information on oxidized oligosialic acid and conjugates are reported. These studies provide a deeper understanding of the conjugation chemistry process and products, which can lead to a better controlled production process.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery.

PubMed

Nam, Joung-Pyo; Kim, Soyoung; Kim, Sung Wan

2018-07-10

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102-128 nm) and positive surface charge (27-34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed better results than poly(CBA-DAH) at 4-fold lower weight ratio. The data of all experiments demonstrate that the synthesized PCDP could be used for efficient gene delivery and could be widely applied. Published by Elsevier B.V.

[35S]-LABELING OF THE SALMONELLA TYPHIMURIUM GLUTATHIONE POOL TO ASSESS GLUTATHIONE-MEDIATED DNA BINDING BY 1,2-DIBROMOETHANE

EPA Science Inventory

Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification m...

Synthesis of surface-anchored DNA-polymer bioconjugates using reversible addition-fragmentation chain transfer polymerization.

PubMed

He, Peng; He, Lin

2009-07-13

We report here an approach to grafting DNA-polymer bioconjugates on a planar solid support using reversible addition-fragmentation chain transfer (RAFT) polymerization. In particular, a trithiocarbonate compound as the RAFT chain transfer agent (CTA) is attached to the distal point of a surface-immobilized oligonucleotide. Initiation of RAFT polymerization leads to controlled growth of polymers atop DNA molecules on the surface. Growth kinetics of poly(monomethoxy-capped oligo(ethylene glycol) methacrylate) atop DNA molecules is investigated by monitoring the change of polymer film thickness as a function of reaction time. The reaction conditions, including the polymerization temperature, the initiator concentration, the CTA surface density, and the selection of monomers, are varied to examine their impacts on the grafting efficiency of DNA-polymer conjugates. Comparing to polymer growth atop small molecules, the experimental results suggest that DNA molecules significantly accelerate polymer growth, which is speculated as a result of the presence of highly charged DNA backbones and purine/pyrimidine moieties surrounding the reaction sites.

Pneumococcal conjugate vaccine: economic issues of the introduction of a new childhood vaccine.

PubMed

Ray, G Thomas

2002-06-01

In February 2000, a pneumococcal conjugate vaccine was licensed for use in the USA. This vaccine has been shown to be effective in reducing pneumococcal disease, and has been recommended for universal use in infants. However, pneumococcal conjugate vaccine is by far the most expensive child vaccine series routinely administered in the USA, alone accounting for over 40% of the total purchase price of vaccines for the recommended childhood schedule. This article reviews the existing efficacy and economic studies of pneumococcal conjugate vaccine and discusses the process by which routine use of pneumococcal conjugate vaccine was introduced and the role economic analysis played in that process. Some of the scientific and funding issues relating to its use in both the industrialized and developing world are also discussed.

Development of a high temperature microbial fermentation process for butanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeor, Jeffery D. St.; Reed, David W.; Daubaras, Dayna L.

2015-08-01

Transforming renewable biomass into cost-competitive high-performance biofuels and bioproducts is key to the U.S. future energy and chemical needs. Butanol production by microbial fermentation for chemical conversion to polyolefins, elastomers, drop-in jet or diesel fuel, and other chemicals is a promising solution. A high temperature fermentation process could decrease energy costs, capital cost, give higher butanol production, and allow for continuous fermentation. In this paper, we describe our approach to genetically transform Geobacillus caldoxylosiliticus, using a pUCG18 plasmid, for potential insertion of a butanol production pathway. Transformation methods tested were electroporation of electrocompetent cells, ternary conjugation with E. coli donormore » and helper strains, and protoplast fusion. These methods have not been successful using the current plasmid. Growth controls show cells survive the various methods tested, suggesting the possibility of transformation inhibition from a DNA restriction modification system in G. caldoxylosiliticus, as reported in the literature.« less

Immobilization and stretching of 5'-pyrene-terminated DNA on carbon film deposited on electron microscope grid.

PubMed

Loukanov, Alexandre; Filipov, Chavdar; Lecheva, Marta; Emin, Saim

2015-11-01

The immobilization and stretching of randomly coiled DNA molecules on hydrophobic carbon film is a challenging microscopic technique, which possess various applications, especially for genome sequencing. In this report the pyrenyl nucleus is used as an anchor moiety to acquire higher affinity of double stranded DNA to the graphite surface. DNA and pyrene are joined through a linker composed of four aliphatic methylene groups. For the preparation of pyrene-terminated DNA a multifunctional phosphoramidite monomer compound was designed. It contains pyrenylbutoxy group as an anchor moiety for π-stacking attachment to the carbon film, 2-cyanoethyloxy, and diisopropylamino as coupling groups for conjugation to activated oligonucleotide chain or DNA molecule. This monomer derivative was suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The successful immobilization and stretching of pyrene-terminated DNA was demonstrated by conventional 100 kV transmission electron microscope. The microscopic analysis confirmed the stretched shape of the negatively charged nucleic acid pieces on the hydrophobic carbon film. © 2015 Wiley Periodicals, Inc.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Mannosylated polyion complexes for in vivo gene delivery into CD11c(+) dendritic cells.

PubMed

Raviv, Lior; Jaron-Mendelson, Michal; David, Ayelet

2015-02-02

Dendritic cells (DCs) possess unique abilities in initiating primary immune responses and thus represent prime targets for DNA-based vaccinations. Here, we describe the design and synthesis of mannosylated polyion complexes (PICs) composed of cationic polyethylenimine (PEI) and hydrophilic polyethylene glycol (PEG) segments, and bearing mono- and trivalent mannose as a ligand for targeting mannose receptor (MR/CD206)-positive DCs. Amino-terminated mannose (Man)-containing ligands in mono- and trivalent presentations (Man- and Man3-, respectively) were prepared and conjugated to PEG via an N-hydroxysuccinimide (NHS)-activated terminal. Thiolated PEI was conjugated to the mannosylated PEG via the maleimide (MAL)-activated terminal. The resulting positively charged diblock copolymers bearing mannoses (Man-PEG-b-PEI and Man3-PEG-b-PEI) were self-assembled with DNA to form PICs with lower surface charge than did their PEI building block and mean hydrodynamic diameters in the range of 100-450 nm, depending on the N/P ratio. Man3-PEG-b-PEI demonstrated a 3-4-fold greater transfection efficiency in MR-positive dendritic cell lines (THP-1, DC2.4), relative to Man-PEG-b-PEI, exhibited low cytotoxicity when compared with PEI, and showed low transfection efficiency in nondendritic HeLa cells. In preliminary in vivo experiments, Man-PEG-b-PEI/DNA and Man3-PEG-b-PEI/DNA demonstrated 2-3-fold higher gene delivery efficiency into CD11c(+) DCs collected from inguinal lymph nodes of C57/BL6 mice, when compared to PEI/DNA complexes, as shown by GFP expression measurements, 24 h post subcutaneous injection. The results indicate that the mannosylated PICs are a safe and effective gene delivery system, showing in vivo specificity toward CD11c(+) DCs.

Glutathione-S-conjugate transport in plants

DOEpatents

Rea, Philip A.; Lu, Yu-Ping; Li, Ze-Sheng

2000-01-01

The invention includes an isolated DNA encoding a plant GS-X pump polypeptide and an isolated preparation of a plant GS-X pump polypeptide. Also included is an isolated preparation of a nucleic acid which is antisense in orientation to a portion or all of a plant GS-X pump gene. The invention also includes a cells, vectors and transgenic plants having an isolated DNA encoding a plant GS-X pump and methods of use thereof. In addition, the invention relates to plant GS-X pump promoter sequences and the uses thereof.

Molecular Approach to Conjugated Polymers with Biomimetic Properties.

PubMed

Baek, Paul; Voorhaar, Lenny; Barker, David; Travas-Sejdic, Jadranka

2018-06-13

The field of bioelectronics involves the fascinating interplay between biology and human-made electronics. Applications such as tissue engineering, biosensing, drug delivery, and wearable electronics require biomimetic materials that can translate the physiological and chemical processes of biological systems, such as organs, tissues. and cells, into electrical signals and vice versa. However, the difference in the physical nature of soft biological elements and rigid electronic materials calls for new conductive or electroactive materials with added biomimetic properties that can bridge the gap. Soft electronics that utilize organic materials, such as conjugated polymers, can bring many important features to bioelectronics. Among the many advantages of conjugated polymers, the ability to modulate the biocompatibility, solubility, functionality, and mechanical properties through side chain engineering can alleviate the issues of mechanical mismatch and provide better interface between the electronics and biological elements. Additionally, conjugated polymers, being both ionically and electrically conductive through reversible doping processes provide means for direct sensing and stimulation of biological processes in cells, tissues, and organs. In this Account, we focus on our recent progress in molecular engineering of conjugated polymers with tunable biomimetic properties, such as biocompatibility, responsiveness, stretchability, self-healing, and adhesion. Our approach is general and versatile, which is based on functionalization of conjugated polymers with long side chains, commonly polymeric or biomolecules. Applications for such materials are wide-ranging, where we have demonstrated conductive, stimuli-responsive antifouling, and cell adhesive biointerfaces that can respond to external stimuli such as temperature, salt concentration, and redox reactions, the processes that in turn modify and reversibly switch the surface properties. Furthermore, utilizing the advantageous chemical, physical, mechanical and functional properties of the grafts, we progressed into grafting of the long side chains onto conjugated polymers in solution, with the vision of synthesizing solution-processable conjugated graft copolymers with biomimetic functionalities. Examples of the developed materials to date include rubbery and adhesive photoluminescent plastics, biomolecule-functionalized electrospun biosensors, thermally and dually responsive photoluminescent conjugated polymers, and tunable self-healing, adhesive, and stretchable strain sensors, advanced functional biocidal polymers, and filtration membranes. As outlined in these examples, the applications of these biomimetic, conjugated polymers are still in the development stage toward truly printable, organic bioelectronic devices. However, in this Account, we advocate that molecular engineering of conjugated polymers is an attractive approach to a versatile class of organic electronics with both ionic and electrical conductivity as well as mechanical properties required for next-generation bioelectronics.

Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

NASA Astrophysics Data System (ADS)

Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

2016-05-01

A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

Antibody-Mediated Small Molecule Detection Using Programmable DNA-Switches.

PubMed

Rossetti, Marianna; Ippodrino, Rudy; Marini, Bruna; Palleschi, Giuseppe; Porchetta, Alessandro

2018-06-13

The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.

An ultrasensitive bio-surrogate for nanoporous filter membrane performance metrology directed towards contamination control in microlithography applications

NASA Astrophysics Data System (ADS)

Ahmad, Farhan; Mish, Barbara; Qiu, Jian; Singh, Amarnauth; Varanasi, Rao; Bedford, Eilidh; Smith, Martin

2016-03-01

Contamination tolerances in semiconductor manufacturing processes have changed dramatically in the past two decades, reaching below 20 nm according to the guidelines of the International Technology Roadmap for Semiconductors. The move to narrower line widths drives the need for innovative filtration technologies that can achieve higher particle/contaminant removal performance resulting in cleaner process fluids. Nanoporous filter membrane metrology tools that have been the workhorse over the past decade are also now reaching limits. For example, nanoparticle (NP) challenge testing is commonly applied for assessing particle retention performance of filter membranes. Factors such as high NP size dispersity, low NP detection sensitivity, and high NP particle-filter affinity impose challenges in characterizing the next generation of nanoporous filter membranes. We report a novel bio-surrogate, 5 nm DNA-dendrimer conjugate for evaluating particle retention performance of nanoporous filter membranes. A technique capable of single molecule detection is employed to detect sparse concentration of conjugate in filter permeate, providing >1000- fold higher detection sensitivity than any existing 5 nm-sized particle enumeration technique. This bio-surrogate also offers narrow size distribution, high stability and chemical tunability. This bio-surrogate can discriminate various sub-15 nm pore-rated nanoporous filter membranes based on their particle retention performance. Due to high bio-surrogate detection sensitivity, a lower challenge concentration of bio-surrogate (as compared to other NPs of this size) can be used for filter testing, providing a better representation of customer applications. This new method should provide better understanding of the next generation filter membranes for removing defect-causing contaminants from lithography processes.

Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads.

PubMed

Puthenveetil, Sujiet; Loganzo, Frank; He, Haiyin; Dirico, Ken; Green, Michael; Teske, Jesse; Musto, Sylvia; Clark, Tracey; Rago, Brian; Koehn, Frank; Veneziale, Robert; Falahaptisheh, Hadi; Han, Xiaogang; Barletta, Frank; Lucas, Judy; Subramanyam, Chakrapani; O'Donnell, Christopher J; Tumey, L Nathan; Sapra, Puja; Gerber, Hans Peter; Ma, Dangshe; Graziani, Edmund I

2016-08-17

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

Competition between conjugation and M13 phage infection in Escherichia coli in the absence of selection pressure: a kinetic study.

PubMed

Wan, Zhenmao; Goddard, Noel L

2012-10-01

Inter- and intraspecies horizontal gene transfer enabled by bacterial secretion systems is a powerful mechanism for bacterial genome plasticity. The type IV secretion system of Escherichia coli, encoded by the F plasmid, enables cell-to-cell contact and subsequent DNA transfer known as conjugation. Conjugation is compromised by phage infection that specifically targets the secretion machinery. Hence, the use of phages to regulate the spread of genes, such as acquired antibiotic resistance or as general biosanitation agents, has gained interest. To predict the potential efficacy, the competition kinetics must first be understood. Using quantitative PCR to enumerate genomic loci in a resource-limited batch culture, we quantify the infection kinetics of the nonlytic phage M13 and its impact on conjugation in the absence of selection pressure (isogenic set). Modeling the resulting experimental data reveals the cellular growth rate to be reduced to 60% upon phage infection. We also find a maximum phage infection rate of 3×10(-11) mL phage(-1) min(-1) which is only 1 order of magnitude slower than the maximum conjugation rate (3×10(-10) mL cell(-1) min(-1)), suggesting phages must be in significant abundance to be effective antagonists to horizontal gene transfer. In the regime where the number of susceptible cells (F(+)) and phages are equal upon initial infection, we observe the spread of the conjugative plasmid throughout the cell population despite phage infection, but only at 10% of the uninfected rate. This has interesting evolutionary implications, as even in the absence of selection pressure, cells maintain the ability to conjugate despite phage vulnerability and the associated growth consequences.

Analysis of Ligand-Receptor Association and Intermediate Transfer Rates in Multienzyme Nanostructures with All-Atom Brownian Dynamics Simulations.

PubMed

Roberts, Christopher C; Chang, Chia-En A

2016-08-25

We present the second-generation GeomBD Brownian dynamics software for determining interenzyme intermediate transfer rates and substrate association rates in biomolecular complexes. Substrate and intermediate association rates for a series of enzymes or biomolecules can be compared between the freely diffusing disorganized configuration and various colocalized or complexed arrangements for kinetic investigation of enhanced intermediate transfer. In addition, enzyme engineering techniques, such as synthetic protein conjugation, can be computationally modeled and analyzed to better understand changes in substrate association relative to native enzymes. Tools are provided to determine nonspecific ligand-receptor association residence times, and to visualize common sites of nonspecific association of substrates on receptor surfaces. To demonstrate features of the software, interenzyme intermediate substrate transfer rate constants are calculated and compared for all-atom models of DNA origami scaffold-bound bienzyme systems of glucose oxidase and horseradish peroxidase. Also, a DNA conjugated horseradish peroxidase enzyme was analyzed for its propensity to increase substrate association rates and substrate local residence times relative to the unmodified enzyme. We also demonstrate the rapid determination and visualization of common sites of nonspecific ligand-receptor association by using HIV-1 protease and an inhibitor, XK263. GeomBD2 accelerates simulations by precomputing van der Waals potential energy grids and electrostatic potential grid maps, and has a flexible and extensible support for all-atom and coarse-grained force fields. Simulation software is written in C++ and utilizes modern parallelization techniques for potential grid preparation and Brownian dynamics simulation processes. Analysis scripts, written in the Python scripting language, are provided for quantitative simulation analysis. GeomBD2 is applicable to the fields of biophysics, bioengineering, and enzymology in both predictive and explanatory roles.

The repABC plasmids with quorum-regulated transfer systems in members of the Rhizobiales divide into two structurally and separately evolving groups

DOE PAGES

Wetzel, Margaret E.; Olsen, Gary J.; Chakravartty, Vandana; ...

2015-11-19

The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from thosemore » coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. Here, we conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function.« less

The repABC plasmids with quorum-regulated transfer systems in members of the Rhizobiales divide into two structurally and separately evolving groups

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetzel, Margaret E.; Olsen, Gary J.; Chakravartty, Vandana

The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from thosemore » coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. Here, we conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function.« less

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer.

PubMed

Kingston, Anthony W; Ponkratz, Christine; Raleigh, Elisabeth A

2017-04-01

Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents r ecombination- p romoting n uclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert. In vitro , RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes. IMPORTANCE Bacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo For RpnA, we demonstrate in vitro action as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching. Copyright © 2017 Kingston et al.

Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

PubMed

Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

2014-06-17

Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

An efficient nonviral gene-delivery vector based on hyperbranched cationic glycogen derivatives.

PubMed

Liang, Xuan; Ren, Xianyue; Liu, Zhenzhen; Liu, Yingliang; Wang, Jue; Wang, Jingnan; Zhang, Li-Ming; Deng, David Yb; Quan, Daping; Yang, Liqun

2014-01-01

The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector. A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino)-1-propylamine (DMAPA-Glyp) and 1-(2-aminoethyl) piperazine (AEPZ-Glyp) residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid-base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA) condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney) and the CNE2 (human nasopharyngeal carcinoma) cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley rats) was evaluated to identify the safety and transfection efficiency. The hyperbranched cationic glycogen derivatives conjugated with DMAPA and AEPZ residues were synthesized. They exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI). They were able to bind and condense pDNA to form the complexes of 100-250 nm in size. The transfection efficiency of the DMAPA-Glyp/pDNA complexes was higher than those of the AEPZ-Glyp/pDNA complexes in both the 293T and CNE2 cells, and almost equal to those of bPEI. Furthermore, pDNA could be more safely delivered to the blood vessels in brain tissue of Sprague Dawley rats by the DMAPA-Glyp derivatives, and then expressed as green fluorescence protein, compared with the control group. The hyperbranched cationic glycogen derivatives, especially the DMAPA-Glyp derivatives, showed high gene-transfection efficiency, good blood compatibility, and low cyto toxicity when transfected in vitro and in vivo, which are novel potential nonviral gene vectors.

Multicellular Computing Using Conjugation for Wiring

PubMed Central

Goñi-Moreno, Angel; Amos, Martyn; de la Cruz, Fernando

2013-01-01

Recent efforts in synthetic biology have focussed on the implementation of logical functions within living cells. One aim is to facilitate both internal “re-programming” and external control of cells, with potential applications in a wide range of domains. However, fundamental limitations on the degree to which single cells may be re-engineered have led to a growth of interest in multicellular systems, in which a “computation” is distributed over a number of different cell types, in a manner analogous to modern computer networks. Within this model, individual cell type perform specific sub-tasks, the results of which are then communicated to other cell types for further processing. The manner in which outputs are communicated is therefore of great significance to the overall success of such a scheme. Previous experiments in distributed cellular computation have used global communication schemes, such as quorum sensing (QS), to implement the “wiring” between cell types. While useful, this method lacks specificity, and limits the amount of information that may be transferred at any one time. We propose an alternative scheme, based on specific cell-cell conjugation. This mechanism allows for the direct transfer of genetic information between bacteria, via circular DNA strands known as plasmids. We design a multi-cellular population that is able to compute, in a distributed fashion, a Boolean XOR function. Through this, we describe a general scheme for distributed logic that works by mixing different strains in a single population; this constitutes an important advantage of our novel approach. Importantly, the amount of genetic information exchanged through conjugation is significantly higher than the amount possible through QS-based communication. We provide full computational modelling and simulation results, using deterministic, stochastic and spatially-explicit methods. These simulations explore the behaviour of one possible conjugation-wired cellular computing system under different conditions, and provide baseline information for future laboratory implementations. PMID:23840385

The SUMO Pathway Is Developmentally Regulated and Required for Programmed DNA Elimination in Paramecium tetraurelia† ‡

PubMed Central

Matsuda, Atsushi; Forney, James D.

2006-01-01

Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling. PMID:16682458

Using inositol as a biocompatible ligand for efficient transgene expression

PubMed Central

Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

2015-01-01

Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

Micronucleus-specific histone H1 is required for micronuclear chromosome integrity in Tetrahymena thermophila

PubMed Central

Qiao, Juxia; Xu, Jing; Bo, Tao

2017-01-01

Histone H1 molecules play a key role in establishing and maintaining higher order chromatin structures. They can bind to linker DNA entering and exiting the nucleosome and regulate transcriptional activity. Tetrahymena thermophila has two histone H1, namely, macronuclear histone H1 and micronuclear histone H1 (Mlh1). Mlh1 is specifically localized at micronuclei during growth and starvation stages. Moreover, Mlh1 is localized around micronuclei and forms a specific structure during the conjugation stage. It co-localizes partially with spindle apparatus during micronuclear meiosis. Analysis of MLH1 knock-out revealed that Mlh1 was required for the micronuclear integrity and development during conjugation stage. Overexpression of Mlh1 led to abnormal conjugation progression. RT-PCR analysis indicated that the expression level of HMGB3 increased in ΔMLH1 strains, while the expression level of MLH1 increased in ΔHMGB3 cells during conjugation. These results indicate that micronuclear integrity and sexual development require normal expression level of Mlh1 and that HmgB3 and Mlh1 may functionally compensate each other in regulating micronuclear structure in T. thermophila. PMID:29095884

Characterization of basic amino acids-conjugated PAMAM dendrimers as gene carriers for human adipose-derived mesenchymal stem cells.

PubMed

Bae, Yoonhee; Lee, Sunray; Green, Eric S; Park, Jung Hyun; Ko, Kyung Soo; Han, Jin; Choi, Joon Sig

2016-03-30

Since mesenchymal stem cells (MSCs) can self-renew and differentiate into multiple cell types, the delivery of genes to this type of cell can be an important tool in the emerging field of tissue regeneration and engineering. However, development of more efficient and safe nonviral vectors for gene delivery to stem cells in particular still remains a great challenge. In this study, we describe a group of nonviral gene delivery vectors, conjugated PAMAM derivatives (PAMAM-H-R, PAMAM-H-K, and PAMAM-H-O), displaying affinity toward human adipose-derived mesenchymal stem cells (AD-MSCs). Transfection efficiency using pDNA encoding for luciferase (Luc) and enhanced green fluorescent protein (EGFP), and cytotoxicity assays were performed in human AD-MSCs. The results show that transfection efficiencies of conjugated PAMAM derivatives are improved significantly compared to native PAMAM dendrimer, and that among PAMAM derivatives, cytotoxicity of PAMAM-H-K and PAMAM-H-O were very low. Also, treatment of human AD-MSCs to polyplex formation in conjugated PAMAM derivatives, their cellular uptake and localization were analyzed by flow cytometry and confocal microscopy. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeting MED1 LxxLL Motifs for Tissue-Selective Treatment of Human Breast Cancer

DTIC Science & Technology

2012-09-01

his colleagues have successfully conjugated malachite green (MG) aptamer to RNA nanoparticles characterized by a three-way junction (3WJ) pRNA motif...nanoparticle harboring malachite green (MG) aptamer, survivin siRNA and folate-DNA/RNA sequence for targeting delivery, using 3WJ-pRNA as scaffolds. Figure

Plasmid transfer by conjugation as a possible route of horizontal gene transfer and recombination in Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Horizontal gene transfer is an important component of evolution and adaptation of bacterial species. Xylella fastidiosa has the ability to incorporate exogenous DNA into its genome by homologous recombination at relatively high rates. This genetic recombination is believed to play a role in adaptati...

Inhibition of murine DNA methyltransferase Dnmt3a by DNA duplexes containing pyrimidine-2(1H)-one.

PubMed

Cherepanova, N A; Zhuze, A L; Gromova, E S

2010-09-01

Here we studied the inhibition of the catalytic domain of Dnmt3a methyltransferase (Dnmt3a-CD) by DNA duplexes containing the mechanism-based inhibitor pyrimidine-2(1H)-one (P) instead of the target cytosine. It has been shown that conjugates of Dnmt3a-CD with P-DNA (DNA containing pyrimidine-2(1H)-one) are not stable to heating at 65°C in 0.1% SDS. The yield of covalent intermediate increases in the presence of the regulatory factor Dnmt3L. The importance of the DNA minor groove for covalent intermediate formation during the methylation reaction catalyzed by Dnmt3a-CD has been revealed. P-DNA was shown to inhibit Dnmt3a-CD; the IC(50) is 830 nM. The competitive mechanism of inhibition of Dnmt3a-CD by P-DNA has been elucidated. It is suggested that therapeutic effect of zebularine could be achieved by inhibition of not only Dnmt1 but also Dnmt3a.

The monomeric form of Neisseria DNA mimic protein DMP19 prevents DNA from binding to the histone-like HU protein

PubMed Central

Ko, Tzu-Ping; Liao, Yi-Ting; Hsu, Kai-Cheng

2017-01-01

DNA mimicry is a direct and effective strategy by which the mimic competes with DNA for the DNA binding sites on other proteins. Until now, only about a dozen proteins have been shown to function via this strategy, including the DNA mimic protein DMP19 from Neisseria meningitides. We have shown previously that DMP19 dimer prevents the operator DNA from binding to the transcription factor NHTF. Here, we provide new evidence that DMP19 monomer can also interact with the Neisseria nucleoid-associated protein HU. Using BS3 crosslinking, gel filtration and isothermal titration calorimetry assays, we found that DMP19 uses its monomeric form to interact with the Neisseria HU dimer. Crosslinking conjugated mass spectrometry was used to investigate the binding mode of DMP19 monomer and HU dimer. Finally, an electrophoretic mobility shift assay (EMSA) confirmed that the DNA binding affinity of HU is affected by DMP19. These results showed that DMP19 is bifunctional in the gene regulation of Neisseria through its variable oligomeric forms. PMID:29220372

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

PubMed

Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

2014-07-01

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

PubMed

Bae, Yun Mi; Kim, Myung Hee; Yu, Gwang Sig; Um, Bong Ho; Park, Hee Kyung; Lee, Hyun-il; Lee, Kang Taek; Suh, Yung Doug; Choi, Joon Sig

2014-02-10

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Multifunctional Surface Modification of Nanodiamonds Based on Dopamine Polymerization.

PubMed

Zeng, Yun; Liu, Wenyan; Wang, Zheyu; Singamaneni, Srikanth; Wang, Risheng

2018-04-03

Surface functionalization of nanodiamonds (NDs), which is of great interest in advanced material and therapeutic applications, requires the immobilization of functional species, such as nucleic acids, bioprobes, drugs, and metal nanoparticles, onto NDs' surfaces to form stable nanoconjugates. However, it is still challenging to modify the surface of NDs due to the complexity of their surface chemistry and the low density of each functional group on the surfaces of NDs. In this work, we demonstrate a general applicable surface functionalization approach for the preparation of ND-based core-shell nanoconjugates using dopamine polymerization. By taking advantage of the universal adhesion and versatile reactivity of polydopamine, we have effectively conjugated DNA and silver nanoparticles onto NDs. Moreover, the catalytic activity of ND-supported silver nanoparticle was characterized by the reduction of 4-nitrophenol, and the addressability of NDs was tested through DNA hybridization that formed satellite ND-gold nanorod conjugation. This simple and robust method we have presented may significantly improve the capability for attaching various functionalities onto NDs and open up new platforms for applications of NDs.

Reducing the background fluorescence in mice receiving fluorophore/inhibitor DNA duplexes.

PubMed

Liang, Minmin; Liu, Xinrong; Liu, Guozheng; Dou, Shuping; Cheng, Dengfeng; Liu, Yuxia; Rusckowski, Mary; Hnatowich, Donald J

2011-02-07

In principle, a DNA duplex consisting of an antisense fluorophore-conjugated major strand hybridized to a shorter complementary inhibitor-conjugated minor strand should provide fluorescence only in the tumor after intravenous administration if designed to remain intact except in the presence in tumor of its mRNA target. While we have obtained impressive tumor images in mice using this approach, there remains some background fluorescence. In this study, tissue homogenates of selected mouse organs were incubated with a test duplex and the kinetics of duplex dissociation in normal tissues were measured. In this manner we were able to identify the liver as the likely major source responsible for the duplex dissociation providing this fluorescence background. Thereafter liver homogenates were used to screen a series of duplex candidates with variable-length minor strands, and dissociation was measured by gel electrophoresis. The selected fluorophore/inhibitor duplex with improved stability displayed an insignificant (P > 0.05) background fluorescence after administration to SKH-1 normal mice and apparently without affecting target mRNA binding in vitro in cell culture or in vivo in tumor bearing mice.

Layered Double Hydroxide Nanotransporter for Molecule Delivery to Intact Plant Cells

PubMed Central

Bao, Wenlong; Wang, Junya; Wang, Qiang; O’Hare, Dermot; Wan, Yinglang

2016-01-01

Here we report a powerful method that facilitates the transport of biologically active materials across the cell wall barrier in plant cells. Positively charged delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) with a 0.5‒2 nm thickness and 30‒60 nm diameter exhibit a high adsorptive capacity for negatively charged biomolecules, including fluorescent dyes such as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate isomer I(FITC) and DNA molecules, forming neutral LDH-nanosheet conjugates. These neutral conjugates can shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Furthermore, typical inhibitors of endocytosis and low temperature incubation did not prevent LDH-lactate-NS internalization, suggesting that LDH-lactate-NS penetrated the plasma membrane via non-endocytic pathways, which will widen the applicability to a variety of plant cells. Moreover, the absence of unwanted side effects in our cytological studies, and the nuclear localization of ssDNA-FITC suggest that nano-LDHs have potential application as a novel gene carrier to plants. PMID:27221055

Folate-conjugated gold nanoparticle as a new nanoplatform for targeted cancer therapy.

PubMed

Samadian, Hadi; Hosseini-Nami, Samira; Kamrava, Seyed Kamran; Ghaznavi, Habib; Shakeri-Zadeh, Ali

2016-11-01

Conventional cancer treatment methods suffer from many limitations such as non-specificity and low efficacy in discrimination between healthy and cancer cells. Recent developments in nanotechnology have introduced novel and smart therapeutic nanomaterials that basically take advantage of various targeting approaches. Targeted nanomaterials selectively bind to the cancer cells and affect them with minor effects on healthy cells. Folic acid (folate) is an essential molecule in DNA synthesis pathway which is highly needed for cancer cell duplication. Some certain cancer cells overexpress folate receptors higher than normal cells, and this fact is the basis of folate targeting strategy. There are many publications reporting various folate conjugated nanomaterials among which folate-conjugated gold nanoparticles hold great promises in targeted cancer therapy. Gold nanoparticles have been identified as promising candidates for new cancer therapy modalities because of biocompatibility, easy synthesis and functionalization, chemo-physical stability, and optical tunable characteristics. In the last decade, there has been a significant explosion in gold nanoparticles research, with a rapid increase in publications related to the area of biomedicine. Although there are many reports published on "gold nanoparticles" and "folate targeting," there are a few reports on "folate-conjugated gold nanoparticles" in biomedical literature. This paper intends to review and illustrate the recent advances in biomedicine which have been designed on the basis of folate-conjugated gold nanoparticles.

Specific gene transfer mediated by galactosylated poly-L-lysine into hepatoma cells.

PubMed

Han, J; Il Yeom, Y

2000-07-20

Plasmid DNA/galactosylated poly-L-lysine(GalPLL) complex was used to transfer luciferase reporter gene in vitro into human hepatoma cells by a receptor-mediated endocytosis process. DNA was combined with galPLL via charge interaction (DNA:GalPLL:fusogenic peptide, 1:0.4:5, w/w/w) and the resulting complex was characterized by dynamic light scattering, gel retardation assay and zeta potential analyzer to determine the particle size, electrostatic charge interaction, and apparent surface charge. The complex was tested for the efficiency of gene transfer in cultured human hepatoblastoma cell line Hep G2 and fibroblast cells NIH/3T3 in vitro. The mean diameter of the complex (DNA:GalPLL=1:0.4, w/w) was 256+/-34.8 nm, and at this ratio, it was positively charged (zeta potential of this complex was 10.1 mV). Hep G2 cells, which express a galactose specific membrane lectin, were efficiently and selectively transfected with the RSV Luc/GalPLL complex in a sugar-dependent manner. NIH/3T3 cells, which do not express the galactose-specific membrane lectin, showed only a marginal level of gene expression. The transfection efficiency of GalPLL-conjugated DNA complex into Hep G2 cells was greatly enhanced in the presence of fusogenic peptide that can disrupt endosomes, where the GalPLL-DNA complex is entrapped with the fusogenic peptide. With the fusogenic peptide KALA, the luciferase activity in Hep G2 cells was ten-fold higher than that of cells transfected in the absence of the fusogenic peptide. Our gene transfer formulation may find potential application for the gene therapy of liver diseases.

Folic acid conjugated mPEG-PEI600 as an efficient non-viral vector for targeted nucleic acid delivery.

PubMed

Xu, Zhenhua; Jin, Jiefu; Siu, Leo K S; Yao, Hong; Sze, Johnny; Sun, Hongzhe; Kung, Hsiang-Fu; Poon, Wai Sang; Ng, Samuel S M; Lin, Marie C

2012-04-15

In this study we describe a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600, and sebacoyl chloride. (1)H NMR, FT-IR, and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. The mPPS-FA/DNA nanoparticles exhibited low cytotoxicity as transfection of B16-F0, U87MG, CHO-1, and Ho-8910 cells produced >80% viability indicating low cytotoxicity of the polymer. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910, and A549 cells was investigated in vitro as compared to the lipid-based transfection agent Lipofectamine2000 and Linear PEI 22 kDa (L-PEI 22 kDa). We found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1, and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be competitively blocked by free folic acid molecules. In contrast, in low FR expressing A549 cells, mPPS-FA showed similar low transfection efficiency as mPPS. Taken together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier. Copyright © 2012 Elsevier B.V. All rights reserved.

Family distribution of anti-F(ab')2 antibodies in relatives of patients with systemic lupus erythematosus.

PubMed Central

Silvestris, F; Searles, R P; Bankhurst, A D; Williams, R C

1985-01-01

Recently we reported an inverse relationship between the levels of anti-F(ab')2 antibodies and disease activity in systemic lupus erythematosus (SLE). The present study focused on anti-F(ab')2 antibodies in unaffected relatives of SLE patients. Sixty sera from first degree family members from 11 SLE families and 49 sera from 8 control families were studied. Percentage of SLE family members with anti-DNA antibodies (15%) was higher than than control family sera (8%, P less than 0.05). Anti-F(ab')2 antibodies were measured using ELISA assays. The SLE family sera had higher amounts of anti-F(ab')2 antibodies than the normal control family group (P = 0.0051). In an effort to determine if anti-F(ab')2 antibodies found in high titres in the sera of some SLE family members had specificity for the F(ab')2 fragment of anti-DNA antibodies of the SLE relative patients, DNA-anti-DNA inhibition experiments were performed using anti-F(ab')2 prepared from the relative in parallel with anti-F(ab')2 prepared from normal controls with equivalent high titres of serum anti-F(ab')2. Inhibition exhibited by anti-F(ab')2 of first degree relatives was higher than that obtained from control normal donors (P less than 0.02). Such differences in inhibition were not recorded using a control tetanus toxoid-anti-tetanus toxoid assay. In direct binding ELISA experiments, peroxidase-conjugated anti-F(ab')2 antibodies from the same first degree relative showed high relative specificity against purified anti-DNA antibodies of his SLE proband when compared to those obtained against different anti-DNA antibodies isolated from unrelated SLE patients (P less than 0.001). Such a substantial difference was not observed in parallel experiments using peroxidase conjugated anti-F(ab')2 antibodies from normal controls unrelated to SLE subjects. PMID:3874025

An Electrochemical DNA Sensing System Using Modified Nanoparticle Probes for Detecting Methicillin-Resistant Staphylococcus aureus.

PubMed

Sakamoto, Hiroaki; Amano, Yoshihisa; Satomura, Takenori; Suye, Shin-Ichiro

2017-01-01

We have developed a novel, highly sensitive, biosensing system for detecting methicillin-resistant Staphylococcus aureus (MRSA). The system employs gold nanoparticles (AuNPs), magnetic nanoparticles (mNPs), and an electrochemical detection method. We have designed and synthesized ferrocene- and single-stranded DNA-conjugated nanoparticles that hybridize to MRSA DNA. Hybridized complexes are easily separated by taking advantage of mNPs. A current response could be obtained through the oxidation of ferrocene on the AuNP surface when a constant potential of +250 mV vs. Ag/AgCl is applied. The enzymatic reaction of L-proline dehydrogenase provides high signal amplification. This sensing system, using a nanoparticle-modified probe, has the ability to detect 10 pM of genomic DNA from MRSA without amplification by the polymerase chain reaction. Current responses are linearly related to the amount of genomic DNA in the range of 10-166 pM. Selectivity is confirmed by demonstrating that this sensing system could distinguish MRSA from Staphylococcus aureus (SA) DNA.

Modelling of DNA-Mediated of Two- and -Three dimensional Protein-Protein and Protein-Nanoparticle Self-Assembly

NASA Astrophysics Data System (ADS)

Millan, Jaime; McMillan, Janet; Brodin, Jeff; Lee, Byeongdu; Mirkin, Chad; Olvera de La Cruz, Monica

Programmable DNA interactions represent a robust scheme to self-assemble a rich variety of tunable superlattices, where intrinsic and in some cases non-desirable nano-scale building blocks interactions are substituted for DNA hybridization events. Recent advances in synthesis has allowed the extension of this successful scheme to proteins, where DNA distribution can be tuned independently of protein shape by selectively addressing surface residues, giving rise to assembly properties in three dimensional protein-nanoparticle superlattices dependent on DNA distribution. In parallel to this advances, we introduced a scalable coarse-grained model that faithfully reproduces the previously observed co-assemblies from nanoparticles and proteins conjugates. Herein, we implement this numerical model to explain the stability of complex protein-nanoparticle binary superlattices and to elucidate experimentally inaccessible features such as protein orientation. Also, we will discuss systematic studies that highlight the role of DNA distribution and sequence on two-dimensional protein-protein and protein-nanoparticle superlattices.

Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions

NASA Astrophysics Data System (ADS)

Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C.; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

2017-02-01

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

PubMed

Limsirichaikul, Siripan; Niimi, Atsuko; Fawcett, Heather; Lehmann, Alan; Yamashita, Shunichi; Ogi, Tomoo

2009-03-01

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

In depth analysis of the quenching of three fluorene-phenylene-based cationic conjugated polyelectrolytes by DNA and DNA bases.

PubMed

Davies, Matthew L; Douglas, Peter; Burrows, Hugh D; Martincigh, Bice; Miguel, Maria da Graça; Scherf, Ullrich; Mallavia, Ricardo; Douglas, Alastair

2014-01-16

The interaction of three cationic poly {9,9-bis[N,N-(trimethylammonium)hexyl]fluorene-co-1,4-phenylene} polymers with average chain lengths of ∼6, 12, and 100 repeat units (PFP-NR36(I),12(Br),100(Br)) with both double and single stranded, short and long, DNA and DNA bases have been studied by steady state and time-resolved fluorescence techniques. Fluorescence of PFP-NR3 polymers is quenched with high efficiency by DNA (both double and single stranded) and DNA bases. The resulting quenching plots are sigmoidal and are not accurately described by using a Stern-Volmer quenching mechanism. Here, the quenching mechanism is well modeled in terms of an equilibrium in which a PFP-NR3/DNA aggregate complex is formed which brings polymer chains into close enough proximity to allow interchain excitation energy migration and quenching at aggregate or DNA base traps. Such an analysis gives equilibrium constants of 8.4 × 10(6) (±1.2 × 10(6)) M(-1) for short-dsDNA and 8.6 × 10(6) (±1.7 × 10(6)) M(-1) for short-ssDNA with PFP-NR36(I).

Effect of oligonucleic acid (ONA) backbone features on assembly of ONA-star polymer conjugates: a coarse-grained molecular simulation study.

PubMed

Condon, Joshua E; Jayaraman, Arthi

2017-10-04

Understanding the impact of incorporating new physical and chemical features in oligomeric DNA mimics, termed generally as "oligonucleic acids" (ONAs), on their structure and thermodynamics will be beneficial in designing novel materials for a variety of applications. In this work, we conduct coarse-grained molecular simulations of ONA-star polymer conjugates with varying ONA backbone flexibility, ONA backbone charge, and number of arms in the star polymer at a constant ONA strand volume fraction to elucidate the effect of these design parameters on the thermodynamics and assembly of multi-arm ONA-star polymer conjugates. We quantify the thermo-reversible behavior of the ONA-star polymer conjugates by quantifying the hybridization of the ONA strands in the system as a function of temperature (i.e. melting curve). Additionally, we characterize the assembly of the ONA-star polymer conjugates by tracking cluster formation and percolation as a function of temperature, as well as cluster size distribution at temperatures near the assembly transition region. The key results are as follows. The melting temperature (T m ) of the ONA strands decreases upon going from a neutral to a charged ONA backbone and upon increasing flexibility of the ONA backbone. Similar behavior is seen for the assembly transition temperature (T a ) with varying ONA backbone charge and flexibility. While the number of arms in the ONA-star polymer conjugate has a negligible effect on the ONA T m in these systems, as the number of ONA-star polymer arms increase, the assembly temperature T a increases and local ordering in the assembled state improves. By understanding how factors like ONA backbone charge, backbone flexibility, and ONA-star polymer conjugate architecture impact the behavior of ONA-star polymer conjugate systems, we can better inform how the selection of ONA chemistry will influence resulting ONA-star polymer assembly.

Excited-State Complexes of Conjugated Polymers: Novel Photophysical Processes and Optoelectronic Materials.

DTIC Science & Technology

1995-03-20

corresponding excited-state complexes were only recently discovered. The results of our extensive studies of intermolecular excimers and exciplexes of many...the luminescence of conjugated polymers. The luminescence and charge photogeneration in exciplexes of conjugated polymers with donor triarylamines will also be presented. jg

Polyethylene glycol and octa-arginine dual-functionalized nanographene oxide: an optimization for efficient nucleic acid delivery.

PubMed

Imani, Rana; Prakash, Satya; Vali, Hojatollah; Faghihi, Shahab

2018-05-29

The successful application of nucleic acid-based therapy for the treatment of various cancers is largely dependent on a safe and efficient delivery system. A dual-functionalized graphene oxide (GO)-based nanocarrier with the conjugation of aminated-polyethylene glycol (PEG-diamine) and octa-arginine (R8) for the intracellular delivery of nucleic acids is proposed. The functionalized sites are covalently co-conjugated and the PEG : R8 molar ratio is optimized at 10 : 1 to achieve a hydrocolloidally stable size of 252 ± 2.0 nm with an effective charge of +40.97 ± 1.05 and an amine-rich content of 10.87 ± 0.4 μmol g-1. The uptake of the nanocarrier in breast cancer cell lines, MCF-7 and MDA-MB 231, is investigated. The siRNA and pDNA condensation ability in the presence and absence of enzymes and the endosomal buffering capacity, as well as the intracellular localization of the gene/nanocarrier complex are also evaluated. Furthermore, the delivery of functional genes associated with the nanocarrier is assessed using c-Myc protein knockdown and EGFP gene expression. The effective uptake of the nanocarrier by the cells shows superior cytocompatibility, and protects the siRNA and pDNA against enzyme degradation while inhibiting their migration with N : P ratios of 10 and 5, respectively. The co-conjugation of PEG-diamine and the cationic cell-penetrating peptide (CPP) into the GO nanocarrier also provides a superior internalization efficacy of 85% in comparison with a commercially available transfection reagent. The c-Myc protein knockdown and EGFP expression, which are induced by the nanocarrier, confirm that the optimized PEG-diamine/R8-functionalized GO could effectively deliver pDNA and siRNA into the cells and interfere with gene expression.

Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3-Helix Bundle-Forming Peptide-PEG Conjugates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lund, Reidar; Ang, JooChuan; Shu, Jessica Y.

Coiled-coil peptide-polymer conjugates are an emerging class of biomaterials. Fundamental understanding of the coiled-coil oligomeric state and assembly process of these hybrid building blocks is necessary to exert control over their assembly into well-defined structures. Here in this paper, we studied the effect of peptide structure and PEGylation on the self-assembly process and oligomeric state of a Langmuir monolayer of amphiphilic coiled-coil peptide-polymer conjugates using X-ray reflectivity (XR) and grazing-incidence X-ray diffraction (GIXD). Our results show that the oligomeric state of PEGylated amphiphiles based on 3-helix bundle-forming peptide is surface pressure dependent, a mixture of dimers and trimers was formedmore » at intermediate surface pressure but transitions into trimers completely upon increasing surface pressure. Moreover, the interhelical distance within the coiled-coil bundle of 3-helix peptide-PEG conjugate amphiphiles was not perturbed under high surface pressure. Present studies provide valuable insights into the self-assembly process of hybrid peptide-polymer conjugates and guidance to develop biomaterials with controlled multivalency of ligand presentation.« less

Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3-Helix Bundle-Forming Peptide-PEG Conjugates

DOE PAGES

Lund, Reidar; Ang, JooChuan; Shu, Jessica Y.; ...

2016-10-26

Coiled-coil peptide-polymer conjugates are an emerging class of biomaterials. Fundamental understanding of the coiled-coil oligomeric state and assembly process of these hybrid building blocks is necessary to exert control over their assembly into well-defined structures. Here in this paper, we studied the effect of peptide structure and PEGylation on the self-assembly process and oligomeric state of a Langmuir monolayer of amphiphilic coiled-coil peptide-polymer conjugates using X-ray reflectivity (XR) and grazing-incidence X-ray diffraction (GIXD). Our results show that the oligomeric state of PEGylated amphiphiles based on 3-helix bundle-forming peptide is surface pressure dependent, a mixture of dimers and trimers was formedmore » at intermediate surface pressure but transitions into trimers completely upon increasing surface pressure. Moreover, the interhelical distance within the coiled-coil bundle of 3-helix peptide-PEG conjugate amphiphiles was not perturbed under high surface pressure. Present studies provide valuable insights into the self-assembly process of hybrid peptide-polymer conjugates and guidance to develop biomaterials with controlled multivalency of ligand presentation.« less

One-step Conjugation of Glycyrrhetinic Acid to Cationic Polymers for High-performance Gene Delivery to Cultured Liver Cell.

PubMed

Cong, Yue; Shi, Bingyang; Lu, Yiqing; Wen, Shihui; Chung, Roger; Jin, Dayong

2016-02-23

Gene therapies represent a promising therapeutic route for liver cancers, but major challenges remain in the design of safe and efficient gene-targeting delivery systems. For example, cationic polymers show good transfection efficiency as gene carriers, but are hindered by cytotoxicity and non-specific targeting. Here we report a versatile method of one-step conjugation of glycyrrhetinic acid (GA) to reduce cytotoxicity and improve the cultured liver cell -targeting capability of cationic polymers. We have explored a series of cationic polymer derivatives by coupling different ratios of GA to polypropylenimine (PPI) dendrimer. These new gene carriers (GA-PPI dendrimer) were systematically characterized by UV-vis,(1)H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, confocal microscopy and flow cytometry. We demonstrate that GA-PPI dendrimers can efficiently load and protect pDNA, via formation of nanostructured GA-PPI/pDNA polyplexes. With optimal GA substitution degree (6.31%), GA-PPI dendrimers deliver higher liver cell transfection efficiency (43.5% vs 22.3%) and lower cytotoxicity (94.3% vs 62.5%, cell viability) than the commercial bench-mark DNA carrier bPEI (25 kDa) with cultured liver model cells (HepG2). There results suggest that our new GA-PPI dendrimer are a promising candidate gene carrier for targeted liver cancer therapy.

One-step Conjugation of Glycyrrhetinic Acid to Cationic Polymers for High-performance Gene Delivery to Cultured Liver Cell

PubMed Central

Cong, Yue; Shi, Bingyang; Lu, Yiqing; Wen, Shihui; Chung, Roger; Jin, Dayong

2016-01-01

Gene therapies represent a promising therapeutic route for liver cancers, but major challenges remain in the design of safe and efficient gene-targeting delivery systems. For example, cationic polymers show good transfection efficiency as gene carriers, but are hindered by cytotoxicity and non-specific targeting. Here we report a versatile method of one-step conjugation of glycyrrhetinic acid (GA) to reduce cytotoxicity and improve the cultured liver cell -targeting capability of cationic polymers. We have explored a series of cationic polymer derivatives by coupling different ratios of GA to polypropylenimine (PPI) dendrimer. These new gene carriers (GA-PPI dendrimer) were systematically characterized by UV-vis,1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, confocal microscopy and flow cytometry. We demonstrate that GA-PPI dendrimers can efficiently load and protect pDNA, via formation of nanostructured GA-PPI/pDNA polyplexes. With optimal GA substitution degree (6.31%), GA-PPI dendrimers deliver higher liver cell transfection efficiency (43.5% vs 22.3%) and lower cytotoxicity (94.3% vs 62.5%, cell viability) than the commercial bench-mark DNA carrier bPEI (25kDa) with cultured liver model cells (HepG2). There results suggest that our new GA-PPI dendrimer are a promising candidate gene carrier for targeted liver cancer therapy. PMID:26902258

Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues.

PubMed

Yang, Bo; Jinnouchi, Akiko; Usui, Kazuteru; Katayama, Tsutomu; Fujii, Masayuki; Suemune, Hiroshi; Aso, Mariko

2015-08-19

We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde. Efficient reductive amination between the DNA-binding domain of Escherichia coli DnaA protein and ODNs carrying OAS in the DnaA-binding sequence proceeded at the lysine residue in proximity to the phosphate group at the 5'-position of the OAS, in contrast to unsuccessful conjugation with abasic site ODNs, even though they have similar aldehydes. Theoretical calculation indicated that the C1-aldehyde of OAS was more accessible to the target lysine than that of the abasic site. These results demonstrate the potential utility of cross-linking strategies that use dicarbonyl-containing ODNs for the study of protein-nucleic acid interactions. Conjugation with a lysine-containing peptide that lacked specific affinity for ODN was also successful, further highlighting the advantages of 1,4-dicarbonyls.

Methylotroph cloning vehicle

DOEpatents

Hanson, Richard S.; Allen, Larry N.

1989-04-25

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C.sub.1 -utilizing host to the C.sub.1 -utilizing host; DNA providing resistance to two antibiotics to which the wild-type C.sub.1 -utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C.sub.1 -utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C.sub.1 -utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C.sub.1 -utilizing (e.g., E. coli) host, and then conjugated with a selected C.sub.1 -utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C.sub.1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.

Theory of end-labeled free-solution electrophoresis: is the end effect important?

PubMed

Chubynsky, Mykyta V; Slater, Gary W

2014-03-01

In the theory of free-solution electrophoresis of a polyelectrolyte (such as the DNA) conjugated with a "drag-tag," the conjugate is divided into segments of equal hydrodynamic friction and its electrophoretic mobility is calculated as a weighted average of the mobilities of individual segments. If all the weights are assumed equal, then for an electrically neutral drag-tag, the elution time t is predicted to depend linearly on the inverse DNA length 1/M. While it is well-known that the equal-weights assumption is approximate and in reality the weights increase toward the ends, this "end effect" has been assumed to be small, since in experiments the t(1/M) dependence seems to be nearly perfectly linear. We challenge this assumption pointing out that some experimental linear fits do not extrapolate to the free (i.e. untagged) DNA elution time in the limit 1/M→0, indicating nonlinearity outside the fitting range. We show that a theory for a flexible polymer taking the end effect into account produces a nonlinear curve that, however, can be fitted with a straight line over a limited range of 1/M typical of experiments, but with a "wrong" intercept, which explains the experimental results without additional assumptions. We also study the influence of the flexibilities of the charged and neutral parts. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pullulan-protamine as efficient haemocompatible gene delivery vector: synthesis and in vitro characterization.

PubMed

Priya, S S; Rekha, M R; Sharma, Chandra P

2014-02-15

Biodegradable non-viral vectors with good transfection efficiency is essential for successful gene delivery. The purpose of this study was to design a non-viral vector by conjugating protamine to pullulan and elucidate the potential use of pullulan protamine conjugate (PPA) as an effective, non toxic and haemocompatible gene delivery system. The particle size and surface charge were measured using Nanosizer. Derivatization was confirmed by NMR, FTIR and DSC analyses. Acid base titration revealed the buffering behaviour of the conjugate. The protection of DNA from nuclease enzyme and interaction of plasma components on the stability of nanoplexes were also analysed. The uptake studies confirmed the plasmid delivery into the nucleus and the inhibitor studies determined the uptake mechanism. Transfection experiments revealed the capability of PPA to cellular uptake in C6 cells and facilitate high gene expression. Thus, PPA proves to be a promising non-viral vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous Iron Is Effective in Multiple Preclinical Cancer Models

PubMed Central

2016-01-01

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics. PMID:27936709

Bioactive ruthenium(II)-arene complexes containing modified 18β-glycyrrhetinic acid ligands.

PubMed

Kong, Yaqiong; Chen, Feng; Su, Zhi; Qian, Yong; Wang, Fang-Xin; Wang, Xiuxiu; Zhao, Jing; Mao, Zong-Wan; Liu, Hong-Ke

2018-05-01

Metal-arene complexes containing bioactive natural-product derived ligands can have new and unusual properties. We report the synthesis, characterization and antiproliferative activity of two new Ru(II) arene complexes with imidazole (dichlorido complex 1) or bipyridyl (chlorido complex 2) ligands conjugated to 18β-glycyrrhetinic acid, an active triterpenoid metabolite of Glycyrrhiza glabra. In general, the conjugated ligands and complexes showed only moderate activity against HeLa (cervical), MCF-7 (breast) and A2780 (ovarian) cancer cells, although the activity of complex 2 in the former two cell lines approached that of the drug cisplatin. Complex 2 (in contrast to complex 1) also exhibited significant activity towards both Gram-positive S. aureus and Gram-negative E. coil bacteria. Complex 2 can induce condensation of DNA and enhances the generation of intracellular reactive oxygen species (ROS). The conjugation of natural products to ligands in organometallic half-sandwich complexes provides a strategy to enhance their biological activities. Copyright © 2018 Elsevier Inc. All rights reserved.

Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens.

PubMed

Hoff, Grégory; Bertrand, Claire; Piotrowski, Emilie; Thibessard, Annabelle; Leblond, Pierre

2017-01-01

Most bacterial organisms rely on homologous recombination to repair DNA double-strand breaks and for the post-replicative repair of DNA single-strand gaps. Homologous recombination can be divided into three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a recA-dependent synaptic step allowing the invasion of an intact copy and the formation of Holliday junctions, and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step. In Escherichia coli, branch migration and resolution are performed by the RuvABC complex, but could also rely on the RecG helicase in a redundant manner. In this study, we show that recG and ruvABC are well-conserved among Streptomyces. ΔruvABC, ΔrecG and ΔruvABC ΔrecG mutant strains were constructed. ΔruvABC ΔrecG is only slightly affected by exposure to DNA damage (UV). We also show that conjugational recombination decreases in the absence of RuvABC and RecG, but that intra-chromosomal recombination is not affected. These data suggest that RuvABC and RecG are indeed involved in homologous recombination in Streptomyces ambofaciens and that alternative factors are able to take over Holliday junction in Streptomyces. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Gold Nanoparticle-Quantum Dot Fluorescent Nanohybrid: Application for Localized Surface Plasmon Resonance-induced Molecular Beacon Ultrasensitive DNA Detection

NASA Astrophysics Data System (ADS)

Adegoke, Oluwasesan; Park, Enoch Y.

2016-11-01

In biosensor design, localized surface plasmon resonance (LSPR)-induced signal from gold nanoparticle (AuNP)-conjugated reporter can produce highly sensitive nanohybrid systems. In order to retain the physicochemical properties of AuNPs upon conjugation, high colloidal stability in aqueous solution is needed. In this work, the colloidal stability with respect to the zeta potential (ZP) of four negatively charged thiol-functionalized AuNPs, thioglycolic (TGA)-AuNPs, 3-mercaptopropionic acid (MPA)-AuNPs, l-cysteine-AuNPs and l-glutathione (GSH)-AuNPs, and a cationic cyteamine-capped AuNPs was studied at various pHs, ionic strength, and NP concentration. A strong dependence of the ZP charge on the nanoparticle (NP) concentration was observed. High colloidal stability was exhibited between pH 3 and 9 for the negatively charged AuNPs and between pH 3 and 7 for the cationic AuNPs. With respect to the ionic strength, high colloidal stability was exhibited at ≤104 μM for TGA-AuNPs, l-cysteine-AuNPs, and GSH-AuNPs, whereas ≤103 μM is recommended for MPA-AuNPs. For the cationic AuNPs, very low ionic strength of ≤10 μM is recommended due to deprotonation at higher concentration. GSH-AuNPs were thereafter bonded to SiO2-functionalized alloyed CdZnSeS/ZnSe1.0S1.3 quantum dots (SiO2-Qdots) to form a plasmon-enhanced AuNP-SiO2-Qdots fluorescent nanohybrid. The AuNP-SiO2-Qdots conjugate was afterward conjugated to a molecular beacon (MB), thus forming an ultrasensitive LSPR-induced SiO2-Qdots-MB biosensor probe that detected a perfect nucleotide DNA sequence at a concentration as low as 10 fg/mL. The limit of detection was 11 fg/mL (1.4 fM) while the biosensor probe efficiently distinguished between single-base mismatch and noncomplementary sequence target.

Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

PubMed Central

2012-01-01

Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins. PMID:23134842

The Mechanism of the Long Noncoding RNA HOTAIR in Breast Cancer

DTIC Science & Technology

2014-10-01

a MS2 RNA aptamer at their 3’ ends to allow purification of the RNA via MS2-Maltose binding protein (MS2-MBP) conjugated to amylose resin (Figure...tethering HOTAIR RNA, tagged with a tandem PP7 and tobramycin-binding aptamer (termed the RAT tag), to DNA and chromatin. We performed PCR with a

Array-Based Discovery of Aptamer Pairs

DTIC Science & Technology

2014-12-11

affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via...standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because...conventional selection methods preferentially yield aptamers that recognize a dominant “hot spot” epitope. Our 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND

Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.

PubMed

DeRocco, Vanessa; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A; Weninger, Keith

2010-11-01

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.

Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445

Expressed sequences tags of the anther smut fungus, Microbotryum violaceum, identify mating and pathogenicity genes

PubMed Central

Yockteng, Roxana; Marthey, Sylvain; Chiapello, Hélène; Gendrault, Annie; Hood, Michael E; Rodolphe, François; Devier, Benjamin; Wincker, Patrick; Dossat, Carole; Giraud, Tatiana

2007-01-01

Background The basidiomycete fungus Microbotryum violaceum is responsible for the anther-smut disease in many plants of the Caryophyllaceae family and is a model in genetics and evolutionary biology. Infection is initiated by dikaryotic hyphae produced after the conjugation of two haploid sporidia of opposite mating type. This study describes M. violaceum ESTs corresponding to nuclear genes expressed during conjugation and early hyphal production. Results A normalized cDNA library generated 24,128 sequences, which were assembled into 7,765 unique genes; 25.2% of them displayed significant similarity to annotated proteins from other organisms, 74.3% a weak similarity to the same set of known proteins, and 0.5% were orphans. We identified putative pheromone receptors and genes that in other fungi are involved in the mating process. We also identified many sequences similar to genes known to be involved in pathogenicity in other fungi. The M. violaceum EST database, MICROBASE, is available on the Web and provides access to the sequences, assembled contigs, annotations and programs to compare similarities against MICROBASE. Conclusion This study provides a basis for cloning the mating type locus, for further investigation of pathogenicity genes in the anther smut fungi, and for comparative genomics. PMID:17692127

Process development of a New Haemophilus influenzae type b conjugate vaccine and the use of mathematical modeling to identify process optimization possibilities.

PubMed

Hamidi, Ahd; Kreeftenberg, Hans; V D Pol, Leo; Ghimire, Saroj; V D Wielen, Luuk A M; Ottens, Marcel

2016-05-01

Vaccination is one of the most successful public health interventions being a cost-effective tool in preventing deaths among young children. The earliest vaccines were developed following empirical methods, creating vaccines by trial and error. New process development tools, for example mathematical modeling, as well as new regulatory initiatives requiring better understanding of both the product and the process are being applied to well-characterized biopharmaceuticals (for example recombinant proteins). The vaccine industry is still running behind in comparison to these industries. A production process for a new Haemophilus influenzae type b (Hib) conjugate vaccine, including related quality control (QC) tests, was developed and transferred to a number of emerging vaccine manufacturers. This contributed to a sustainable global supply of affordable Hib conjugate vaccines, as illustrated by the market launch of the first Hib vaccine based on this technology in 2007 and concomitant price reduction of Hib vaccines. This paper describes the development approach followed for this Hib conjugate vaccine as well as the mathematical modeling tool applied recently in order to indicate options for further improvements of the initial Hib process. The strategy followed during the process development of this Hib conjugate vaccine was a targeted and integrated approach based on prior knowledge and experience with similar products using multi-disciplinary expertise. Mathematical modeling was used to develop a predictive model for the initial Hib process (the 'baseline' model) as well as an 'optimized' model, by proposing a number of process changes which could lead to further reduction in price. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:568-580, 2016. © 2016 American Institute of Chemical Engineers.

Morphological Priming by Itself: A Study of Portuguese Conjugations

ERIC Educational Resources Information Center

Verissimo, Joao; Clahsen, Harald

2009-01-01

Does the language processing system make use of abstract grammatical categories and representations that are not directly visible from the surface form of a linguistic expression? This study examines stem-formation processes and conjugation classes, a case of "pure" morphology that provides insight into the role of grammatical structure in…

A novel fluorescent DNA sensor for ultrasensitive detection of Helicobacter pylori.

PubMed

Liu, Ziping; Su, Xingguang

2017-01-15

In this work, a novel fluorescent DNA sensor for ultrasensitive detection of Helicobacter pylori (H. pylori) DNA was developed. This strategy took advantage of DNA hybridization between single-stranded DNA (ssDNA, which had been designed as an aptamer specific for H. pylori DNA) and the complementary target H. pylori DNA, and the feature that ssDNA bound to graphene oxide (GO) with significantly higher affinity than double-stranded DNA (dsDNA). ssDNA were firstly covalent conjugated with CuInS 2 quantum dots (QDs) by reaction between the carboxy group of QDs and amino group modified ssDNA, forming ssDNA-QDs genosensor. In the absence of the complementary target H. pylori DNA, GO could adsorb ssDNA-QDs DNA sensor and efficiently quench the fluorescence of ssDNA-QDs. While the complementary target H. pylori DNA was introduced, the ssDNA-QDs preferentially bound with the H. pylori DNA. The formation of dsDNA would alter the conformation of ssDNA and disturb the interaction between ssDNA and GO. Thus, the dsDNA-QDs/GO system exhibited a stronger fluorescence emission than that of the ssDNA-QDs/GO system. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 and the concentration of H. pylori DNA in the range of 1.25-875pmolL -1 with a detection limit of 0.46pmolL -1 . The proposed method was applied to the determination of H. pylori DNA sequence in milk samples with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel technology for the detection, enrichment, and separation of trace amounts of target DNA based on amino-modified fluorescent magnetic composite nanoparticles.

PubMed

Wang, Guannan; Su, Xingguang

2010-06-01

A novel, highly sensitive technology for the detection, enrichment, and separation of trace amounts of target DNA was developed on the basis of amino-modified fluorescent magnetic composite nanoparticles (AFMN). In this study, the positively charged amino-modified composite nanoparticles conjugate with the negatively charged capture DNA through electrostatic binding. The optimal combination of AFMN and capture DNA was measured by dynamic light scattering (DLS) and UV-vis absorption spectroscopy. The highly sensitive detection of trace amounts of target DNA was achieved through enrichment by means of AFMN. The detection limit for target DNA is 0.4 pM, which could be further improved by using a more powerful magnet. Because of their different melting temperatures, single-base mismatched target DNA could be separated from perfectly complementary target DNA. In addition, the photoluminescence (PL) signals of perfectly complementary target DNA and single-base mismatched DNA as well as the hybridization kinetics of different concentrations of target DNA at different reaction times have also been studied. Most importantly, the detection, enrichment, and separation ability of AFMN was further verified with milk. Simple and satisfactory results were obtained, which show the great potential in the fields of mutation identification and clinical diagnosis.

Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Novel Carriers for DNA, shRNA and siRNA

PubMed Central

Arima, Hidetoshi; Motoyama, Keiichi; Higashi, Taishi

2012-01-01

Gene, short hairpin RNA (shRNA) and small interfering RNA (siRNA) delivery can be particularly used for the treatment of diseases by the entry of genetic materials mammalian cells either to express new proteins or to suppress the expression of proteins, respectively. Polyamidoamine (PAMAM) StarburstTM dendrimers are used as non-viral vectors (carriers) for gene, shRNA and siRNA delivery. Recently, multifunctional PAMAM dendrimers can be used for the wide range of biomedical applications including intracellular delivery of genes and nucleic acid drugs. In this context, this review paper provides the recent findings on PAMAM dendrimer conjugates with cyclodextrins (CyDs) for gene, shRNA and siRNA delivery. PMID:24300184

Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards.

PubMed

O'Driscoll, Jonathan; Glynn, Frances; Fitzgerald, Gerald F; van Sinderen, Douwe

2006-09-01

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.

PubMed

Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A; Schmid, Michael C; Schröder, Gunnar; Vergunst, Annette C; Carena, Ilaria; Dehio, Christoph

2005-01-18

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

The novel conjugative transposon tn1207.3 carries the macrolide efflux gene mef(A) in Streptococcus pyogenes.

PubMed

Santagati, Maria; Iannelli, Francesco; Cascone, Carmela; Campanile, Floriana; Oggioni, Marco R; Stefani, Stefania; Pozzi, Gianni

2003-01-01

The macrolide efflux gene mef(A) of the Streptococcus pyogenes clinical strain 2812A was found to be carried by a 52-kb chromosomal genetic element that could be transferred by conjugation to the chromosome of other streptococcal species. The characteristics of this genetic element are typical of conjugative transposons and was named Tn1207.3. The size of Tn1207.3 was established by pulsed-field gel electrophoresis (PFGE), and DNA sequencing analysis showed that the 7,244 bp at the left end of Tn1207.3 were identical to those of the pneumococcal Tn1207.1 element. Tn1207.3-like genetic elements were found to be inserted at a single specific chromosomal site in 12 different clinical isolates S. pyogenes exhibiting the M phenotype of resistance to macrolides and carrying the mef(A) gene. Tn1207.3 was transferred from S. pyogenes 2812A to Streptococcus pneumoniae, and sequence analysis carried out on six independent transconjugants showed that insertion of Tn1207.3 in the pneumococcal genome always occurred at a single specific site as in Tn1207.1. Using MF2, a representative S. pneumoniae transconjugant, as a donor, Tn1207.3 was transferred again by conjugation to S. pyogenes and Streptococcus gordonii. The previously described nonconjugative element Tn1207.1 of S. pneumoniae appears to be a defective element, part of a longer conjugative transposon that carries mef(A) and is found in clinical isolates of S. pyogenes.

Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling.

PubMed

Fang, Ge-Min; Seitz, Oliver

2017-12-24

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

NASA Astrophysics Data System (ADS)

Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

2016-12-01

A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

PubMed

Zimmermann, Gunther; Li, Yizhou; Rieder, Ulrike; Mattarella, Martin; Neri, Dario; Scheuermann, Jörg

2017-05-04

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantum-dot-based quantitative identification of pathogens in complex mixture

NASA Astrophysics Data System (ADS)

Lim, Sun Hee; Bestwater, Felix; Buchy, Philippe; Mardy, Sek; Yu, Alexey Dan Chin

2010-02-01

In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.

Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine.

PubMed

An, Jeung Hee; Lee, Kwon-Jai; Choi, Jeong-Woo

2016-02-01

Nanotechnology-based bio-barcode amplification analysis offers an innovative approach for detecting neurotransmitters. We evaluated the efficacy of this method for detecting norepinephrine in normal and oxidative-stress damaged dopaminergic cells. Our approach use a combination of DNA barcodes and bead-based immunoassays for detecting neurotransmitters with surface-enhanced Raman spectroscopy (SERS), and provides polymerase chain reaction (PCR)-like sensitivity. This method relies on magnetic Dynabeads containing antibodies and nanoparticles that are loaded both with DNA barcords and with antibodies that can sandwich the target protein captured by the Dynabead-bound antibodies. The aggregate sandwich structures are magnetically separated from the solution and treated to remove the conjugated barcode DNA. The DNA barcodes are then identified by SERS and PCR analysis. The concentration of norepinephrine in dopaminergic cells can be readily detected using the bio-barcode assay, which is a rapid, high-throughput screening tool for detecting neurotransmitters.

Metabolism and toxicity of arsenicals in mammals.

PubMed

Sattar, Adeel; Xie, Shuyu; Hafeez, Mian Abdul; Wang, Xu; Hussain, Hafiz Iftikhar; Iqbal, Zahid; Pan, Yuanhu; Iqbal, Mujahid; Shabbir, Muhammad Abubakr; Yuan, Zonghui

2016-12-01

Arsenic (As) is a metalloid usually found in organic and inorganic forms with different oxidation states, while inorganic form (arsenite As-III and arsenate As-v) is considered to be more hazardous as compared to organic form (methylarsonate and dimethylarsinate), with mild or no toxicity in mammals. Due to an increasing trend to using arsenicals as growth promoters or for treatment purposes, the understanding of metabolism and toxicity of As gets vital importance. Its toxicity is mainly depends on oxi-reduction states (As-III or As-v) and the level of methylation during the metabolism process. Currently, the exact metabolic pathways of As have yet to be confirmed in humans and food producing animals. Oxidative methylation and glutathione conjugation is believed to be major pathways of As metabolism. Oxidative methylation is based on conversion of Arsenite in to mono-methylarsonic acid and di-methylarsenic acid in mammals. It has been confirmed that As is only methylated in the presence of glutathione or thiol compounds, suggesting that As is being methylated in trivalent states. Subsequently, non-conjugated trivalent arsenicals are highly reactive with thiol which converts the trivalent arsenicals in to less toxic pentavalent forms. The glutathione conjugate stability of As is the most important factor for determining the toxicity. It can lead to DNA damage by alerting enzyme profile and production of reactive oxygen and nitrogen species which causes the oxidative stress. Moreover, As causes immune-dysfunction by hindering cellular and humeral immune response. The present review discussed different metabolic pathways and toxic outcomes of arsenicals in mammals which will be helpful in health risk assessment and its impact on biological world. Copyright © 2016. Published by Elsevier B.V.

Molecular markers of trichloroethylene-induced toxicity in human kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lash, Lawrence H.; Putt, David A.; Hueni, Sarah E.

Difficulties in evaluation of trichloroethylene (TRI)-induced toxicity in humans and extrapolation of data from laboratory animals to humans are due to the existence of multiple target organs, multiple metabolic pathways, sex-, species-, and strain-dependent differences in both metabolism and susceptibility to toxicity, and the lack or minimal amount of human data for many target organs. The use of human tissue for mechanistic studies is thus distinctly advantageous. The kidneys are one target organ for TRI and metabolism by the glutathione (GSH) conjugation pathway is responsible for nephrotoxicity. The GSH conjugate is processed further to produce the cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC),more » which is the penultimate nephrotoxic species. Confluent, primary cultures of human proximal tubular (hPT) cells were used as the model system. Although cells in log-phase growth, which are undergoing more rapid DNA synthesis, would give lower LD{sub 50} values, confluent cells more closely mimic the in vivo proximal tubule. DCVC caused cellular necrosis only at relatively high doses (>100 {mu}M) and long incubation times (>24 h). In contrast, both apoptosis and enhanced cellular proliferation occurred at relatively low doses (10-100 {mu}M) and early incubation times (2-8 h). These responses were associated with prominent changes in expression of several proteins that regulate apoptosis (Bcl-2, Bax, Apaf-1, Caspase-9 cleavage, PARP cleavage) and cellular growth, differentiation and stress response (p53, Hsp27, NF-{kappa}B). Effects on p53 and Hsp27 implicate function of protein kinase C, the mitogen activated protein kinase pathway, and the cytoskeleton. The precise pattern of expression of these and other proteins can thus serve as molecular markers for TRI exposure and effect in human kidney.« less

Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation.

PubMed

Vedantam, Gayatri; Knopf, Sarah; Hecht, David W

2006-01-01

Tn5520 is the smallest known bacterial mobilizable transposon and was isolated from an antibiotic resistant Bacteroides fragilis clinical isolate. When a conjugation apparatus is provided in trans, Tn5520 is mobilized (transferred) efficiently within, and from, both Bacteroides spp. and Escherichia coli. Only two genes are present on Tn5520; one encodes an integrase, and the other a multifunctional mobilization (Mob) protein BmpH. BmpH is essential for Tn5520 mobility. The focus of this study was to identify the Tn5520 origin of conjugative transfer (oriT) and to study BmpH-oriT binding. We delimited the functional Tn5520 oriT to a 71 bp sequence upstream of the bmpH gene. A plasmid vector harbouring this minimal 71 bp oriT was mobilized at the same frequency as that of intact Tn5520. The minimal oriT contains one 17 bp inverted repeat (IR) sequence. We constructed and tested multiple IR mutants and showed that the IR was essential in its entirety for mobilization. A nick site sequence (5'-GCTAC-3') was also identified within the minimal oriT; this sequence resembled nick sites found in plasmids of Gram positive origin. We further showed that mutation of a highly conserved GC dinucleotide in the nick site sequence completely abolished mobilization. We also purified BmpH and showed that it specifically bound a Tn5520 oriT fragment in electrophoretic mobility shift assays. We also identified non-nick site sequences within the minimal oriT that were essential for mobilization. We hypothesize that transposon-based single Mob protein systems may contribute to efficient gene dissemination from Bacteroides spp., because fewer DNA processing proteins are required for relaxosome formation.

Identification of an unintended consequence of Nrf2-directed cytoprotection against a key tobacco carcinogen plus a counteracting chemopreventive intervention

PubMed Central

Paonessa, Joseph D.; Ding, Yi; Randall, Kristen L.; Munday, Rex; Argoti, Dayana; Vouros, Paul; Zhang, Yuesheng

2011-01-01

Nrf2 is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. While Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2+/+ mice than in Nrf2−/− mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. While glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, while higher liver UGT activity may protect the liver against ABP it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further demonstrate that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues, but does not appear to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2. PMID:21487034

A Monofunctional Platinum Complex Coordinated to a Rhodium Metalloinsertor Selectively Binds Mismatched DNA in the Minor Groove

PubMed Central

Weidmann, Alyson G.; Barton, Jacqueline K.

2015-01-01

We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh—O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA non-classically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors. PMID:26397309

Solid lipid nanoparticles mediate non-viral delivery of plasmid DNA to dendritic cells

NASA Astrophysics Data System (ADS)

Penumarthi, Alekhya; Parashar, Deepti; Abraham, Amanda N.; Dekiwadia, Chaitali; Macreadie, Ian; Shukla, Ravi; Smooker, Peter M.

2017-06-01

There is an increasing demand for novel DNA vaccine delivery systems, mainly for the non-viral type as they are considered relatively safe. Therefore, solid lipid nanoparticles (SLNs) were investigated for their suitability as a non-viral DNA vaccine delivery system. SLNs were synthesised by a modified solvent-emulsification method in order to study their potential to conjugate with plasmid DNA and deliver them in vitro to dendritic cells using eGFP as the reporter plasmid. The DNA-SLN complexes were characterised by electron microscopy, gel retardation assays and dynamic light scattering. The cytotoxicity assay data supported their biocompatibility and was used to estimate safe threshold concentration resulting in high transfection rate. The transfection efficiency of these complexes in a dendritic cell line was shown to increase significantly compared to plasmid alone, and was comparable to that mediated by lipofectamine. Transmission electron microscopy studies delineated the pathway of cellular uptake. Endosomal escape was observed supporting the mechanism of transfection.

Detection of tyrosine hydroxylase in dopaminergic neuron cell using gold nanoparticles-based barcode DNA.

PubMed

An, Jeung Hee; Oh, Byung-Keun; Choi, Jeong Woo

2013-04-01

Tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosysthesis, is predominantly expressed in several cell groups within the brain, including the dopaminergic neurons of the substantia nigra and ventral tegmental area. We evaluated the efficacy of this protein-detection method in detecting tyrosine hydroxylase in normal and oxidative stress damaged dopaminergic cells. In this study, a coupling of DNA barcode and bead-based immnunoassay for detecting tyrosine hydroxylaser with PCR-like sensitivity is reported. The method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes were identified by PCR analysis. The concentration of tyrosine hydroxylase in dopaminergic cell can be easily and rapidly detected using bio-barcode assay. The bio-barcode assay is a rapid and high-throughput screening tool to detect of neurotransmitter such as dopamine.

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

A monofunctional platinum complex coordinated to a rhodium metalloinsertor selectively binds mismatched DNA in the minor groove.

PubMed

Weidmann, Alyson G; Barton, Jacqueline K

2015-10-05

We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh-O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA nonclassically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and it triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors.

Control of electrochemical signals from quantum dots conjugated to organic materials by using DNA structure in an analog logic gate.

PubMed

Chen, Qi; Yoo, Si-Youl; Chung, Yong-Ho; Lee, Ji-Young; Min, Junhong; Choi, Jeong-Woo

2016-10-01

Various bio-logic gates have been studied intensively to overcome the rigidity of single-function silicon-based logic devices arising from combinations of various gates. Here, a simple control tool using electrochemical signals from quantum dots (QDs) was constructed using DNA and organic materials for multiple logic functions. The electrochemical redox current generated from QDs was controlled by the DNA structure. DNA structure, in turn, was dependent on the components (organic materials) and the input signal (pH). Independent electrochemical signals from two different logic units containing QDs were merged into a single analog-type logic gate, which was controlled by two inputs. We applied this electrochemical biodevice to a simple logic system and achieved various logic functions from the controlled pH input sets. This could be further improved by choosing QDs, ionic conditions, or DNA sequences. This research provides a feasible method for fabricating an artificial intelligence system. Copyright © 2016 Elsevier B.V. All rights reserved.

Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16▿

PubMed Central

Timmery, Sophie; Modrie, Pauline; Minet, Olivier; Mahillon, Jacques

2009-01-01

Conjugation, mobilization, and retromobilization are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in gram-negative species, where retromobilization, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient followed by the retrotransfer of the mobilizable plasmid to the donor. This successive model was established for gram-negative bacteria but was lacking experimental data from the gram-positive counterparts. In the present work, the mobilization and retromobilization abilities of the conjugative plasmid pXO16 from Bacillus thuringiensis subsp. israelensis were studied using the mobilizable plasmids pUB110 and pE194 and the “nonmobilizable” element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model, since different retromobilization frequencies were observed between the small plasmids. More importantly, retromobilization was shown to be delayed by 50 and 150 min for pUB110 and pE194, respectively, compared to pXO16 conjugation. Natural liquid foods (cow milk, soy milk, and rice milk) were used to evaluate the putative ecological impact of these transfers. In cow and soy milk, conjugation, mobilization, and retromobilization were shown to occur at frequencies of 8.0 × 10−1, 1.0 × 10−2, and 1.2 × 10−4 transconjugants per recipient, respectively. These data are comparable to those obtained with LB medium and about 10-fold lower than in the case of rice milk. Taken together, these results emphasize the potential role of plasmid capture played by B. thuringiensis in natural environments. PMID:19181805

Toward the in vivo study of captopril-conjugated quantum dots

NASA Astrophysics Data System (ADS)

Manabe, Noriyoshi; Hoshino, Akiyoshi; Liang, Yi-qiang; Goto, Tomomasa; Kato, Norihiro; Yamamoto, Kenji

2005-04-01

Photo-luminescent semiconductor quantum dots are nanometer-size probes that have the potential to be applied to the fields of the bio-imaging and the study of the cell mobility inside the body. At the same time, on the other hand, quantum dots are expected to carry some kind of molecules to the local organ inside of the animal body, which leads to the expectation that they can be used as a medicine-carrier. For this purpose, we conjugate (2S)-1-[(2s)-2-Methyl-3-sulfanylpropionyl]pyrrolidine-2-carboxylic acid (cap) with the quantum dot. Cap has the effect as an anti-hypertension drug, which inhibits angiotensin 1 converting enzyme. We conjugated the quantum dot with cap by the exchange reaction avoiding the regions which holds medicinal effect. Quantum dot conjugated with cap (QD-cap) were 3-times brighter than thioglycerol-coated quantum dots (QD-OH). The particle size of cap was 1.1nm and that of QD-cap was 12nm. QD-cap was permeated into the HeLa cells, while QD-MUA were taken into the HeLa cells by endocytosis. In addition, no apoptosis was detected against the cells that permeated QD-cap, because there was no damage to DNA. These results indicated that QD-conjugated medicines (QD-medicine) could be safe in the experiment on the level of the cell. More over, when QD-cap was intravenously injected into Stroke-prone Spontaneously Hypertensive Rats (SHRSP), they reduced blood pressure at systole. Therefore, the anti-hypertension effect of cap remained after conjugated with the quantum dot. These results suggested that QD-medicine were effective on the animal level.

A Review of Therapeutic Aptamer Conjugates with Emphasis on New Approaches

PubMed Central

Bruno, John G.

2013-01-01

The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q) to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA) or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies. PMID:24276022

Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Daehwan; Yeom, Ji-Hyun; Lee, Boeun

The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation andmore » invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer. - Highlights: • TM-JM1/2 peptides are efficiently delivered into cells by AuNP-Apt-conjugates. • TM-JM1/2 peptides loaded onto AuNP-Apt conjugates inhibit DDR2 activation. • Inhibition of DDR2 activation by TM-JM1/2 peptides decreases tumor progression.« less

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

PubMed

Haskett, Timothy L; Terpolilli, Jason J; Ramachandran, Vinoy K; Verdonk, Callum J; Poole, Phillip S; O'Hara, Graham W; Ramsay, Joshua P

2018-03-01

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

Conjugal transferring of resistance gene ptr for improvement of pristinamycin-producing Streptomyces pristinaespiralis.

PubMed

Jin, Zhihua; Jin, Xin; Jin, Qingchao

2010-03-01

Improving pristinamycin production from Streptomyces pristinaespiralis was performed by introducing the resistance gene ptr followed by selection for enhanced tolerance to pristinamycin and fermentation test. To transfer ptr into S. pristinaespiralis, an effective method was established for the first time by using the intergeneric conjugation of DNA from Escherichia coli to S. pristinaespiralis. The procedure was optimized with heat treatment, spore concentration, optimum medium used in conjugation, concentration of MgCl(2), etc. With the optimized conditions, the conjugation frequency was up to 1.36 x 10(-3) exconjugants per recipient. The procedure was used to transfer the ptr gene into S. pristinaespiralis, resulting in 146 exconjugants. These exconjugants were screened on the pristinamycin-resistant plates, and then the fermentation test subsequently. Finally, two strains (SPR1 and SPR2) were obtained with a high yield of 0.11 and 0.15 g/l, respectively, which is about six to eight times more than that of wild-strain ATCC25486. The subculture experiments indicated that the hereditary character of the high-producing S. pristinaespiralis SPR1 and SPR2 was stable. Our work suggests that introducing resistance gene ptr into S. pristinaespiralis could be the way to improve the production of pristinamycin through the enhancement of antibiotic tolerance.

Easy detection of multiple Alexandrium species using DNA chromatography chip.

PubMed

Nagai, Satoshi; Miyamoto, Shigehiko; Ino, Keita; Tajimi, Seisuke; Nishi, Hiromi; Tomono, Jun

2016-01-01

In this study, the Kaneka DNA chromatography chip (KDCC) for the Alexandrium species was successfully developed for simultaneous detection of five Alexandrium species. This method utilizes a DNA-DNA hybridization technology. In the PCR process, specifically designed tagged-primers are used, i.e. a forward primer consisting of a tag domain, which can conjugate with gold nanocolloids on the chip, and a primer domain, which can anneal/amplify the target sequence. However, the reverse primer consists of a tag domain, which can hybridize to the solid-phased capture probe on the chip, and a primer domain, which can anneal/amplify the target sequence. As a result, a red line that originates from gold nanocolloids appears as a positive signal on the chip, and the amplicon is detected visually by the naked eye. This technique is simple, because it is possible to visually detect the target species soon after (<5min) the application of 2μL of PCR amplicon and 65μL of development buffer to the sample pad of the chip. Further, this technique is relatively inexpensive and does not require expensive laboratory equipment, such as real-time Q-PCR machines or DNA microarray detectors, but a thermal cycler. Regarding the detection limit of KDCC for the five Alexandrium species, it varied among species and it was <0.1-10pg and equivalent to 5-500 copies of rRNA genes, indicating that the technique is sensitive enough for practical use to detect several cells of the target species from 1L of seawater. The detection sensitivity of KDCC was also evaluated with two different techniques, i.e. a multiplex-PCR and a digital DNA hybridization by digital DNA chip analyzer (DDCA), using natural plankton assemblages. There was no significant difference in the detection sensitivity among the three techniques, suggesting KDCC can be readily used to monitor the HAB species. Copyright © 2015 Elsevier B.V. All rights reserved.

Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization.

PubMed

Cissell, Kyle A; Rahimi, Yasmeen; Shrestha, Suresh; Deo, Sapna K

2009-01-01

Reassembly of split reporter proteins, also referred to as protein complementation, is utilized in the detection of protein-protein or protein-nucleic acid interactions. In this strategy, a reporter protein is fragmented into two inactive polypeptides to which interacting/binding partners are fused. The interaction between fused partners leads to the formation of a reassembled, active reporter. In this Communication, we have presented a proof-of-concept for the detection of a target nucleic acid sequence based on the reassembly of the bioluminescent reporter Renilla luciferase (Rluc), which is driven by DNA hybridization. Although, reassembly of Rluc though protein interactions has been demonstrated by others, the Rluc reassembly through DNA hybridization has not been shown yet, which is the novelty of this work. It is well established that bioluminescence detection offers significant advantages due to the absence of any background signal. In our study, two rationally designed fragments of Rluc were conjugated to complementary oligonucleotide probes. Hybridization of the two probes with fused Rluc fragments resulted in the reassembly of the fragments, generating active Rluc, measurable by the intensity of light given off upon addition of coelenterazine. Our study also shows that the reassembly of Rluc can be inhibited by an oligonucleotide probe that competes to bind to the hybridized probe-Rluc fragment complex, indicating a potential strategy for the quantitative detection of target nucleic acid. We were able to achieve the reassembly of Rluc fused to oligonucleotide probes using femtomole amounts of the probe-fragment protein conjugate. This concentration is approximately 4 orders of magnitude less than that reported using green fluorescent protein (GFP) as the reporter. A DNA-driven Rluc reassembly study performed in a cellular matrix did not show any interference from the matrix.

Detection of endogenous DNA adducts, O-carboxymethyl-2'-deoxyguanosine and 3-ethanesulfonic acid-2'-deoxycytidine, in the rat stomach after duodenal reflux.

PubMed

Terasaki, Masaru; Totsuka, Yukari; Nishimura, Koichi; Mukaisho, Ken-Ichi; Chen, Kuan-Hao; Hattori, Takanori; Takamura-Enya, Takeji; Sugimura, Takashi; Wakabayashi, Keiji

2008-09-01

The endogenous DNA adducts O(6)-carboxymethyl-deoxyguanosine (O(6)-CM-dG) and 3-ethanesulfonic acid-deoxycytidine (3-ESA-dC) are produced from N-nitroso bile acid conjugates, such as N-nitrosoglycocholic acid (NO-GCA) and N-nitrosotaurocholic acid (NO-TCA), respectively. Formation of these DNA adducts in vivo was here analyzed by 32P-postlabeling in the glandular stomach of rats subjected to duodenal content reflux surgery. In this model, all duodenal contents, including bile acid conjugates, flow back from the jejunum into the gastric corpus. The levels of O(6)-CM-dG found at 4 and 8 weeks after surgery were 40.9 +/- 9.4 and 56.3 +/- 3.2 per 10(8) nucleotides, respectively, whereas the sham operation groups had values of 5.8 +/- 2.3 and 5.9 +/- 0.5 per 10(8) nucleotides. Moreover, adduct spots corresponding to 3-ESA-dC were detected in both duodenal reflux and sham operation groups and levels in the duodenal reflux groups were around four-fold elevated at 11.2 +/- 1.0 and 8.9 +/- 1.0 per 10(8) nucleotides after 4 and 8 weeks, respectively. When the duodenal reflux animals were treated with a nitrite trapping agent, thiazolidine- 4-carboxylic acid (thioproline, TPRO), the levels of O(6)-CM-dG and 3-ESA-dC were reduced to the same levels as in the sham operation animals. These observations suggest that NO-TCA and NO-GCA are formed by nitrosation of glycocholic acid and taurocholic acid, respectively, and these nitroso compounds produce DNA adducts in the glandular stomach of rats subjected to duodenal content reflux surgery.

An ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated lambda exonuclease cleavage-induced signal amplification.

PubMed

Cui, Lin; Li, Yueying; Lu, Mengfei; Tang, Bo; Zhang, Chun-Yang

2018-01-15

Polynucleotide kinase (PNK) plays an essential role in cellular nucleic acid metabolism and the cellular response to DNA damage. However, conventional methods for PNK assay suffer from low sensitivity and involve multiple steps. Herein, we develop a simply electrochemical method for sensitive detection of PNK activity on the basis of Au nanoparticle (AuNP)-mediated lambda exonuclease cleavage-induced signal amplification. We use [Ru(NH 3 ) 6 ] 3+ as the electrochemically active indicator and design two DNA strands (i.e., strand 1 and strand 2) to sense PNK. The assembly of strand 2 on the AuNP surface leads to the formation of AuNP-strand 2 conjugates which can be subsequently immobilized on the gold electrode through the hybridization of strand 1 with strand 2 for the generation of a high electrochemical signal. The presence of PNK induces the phosphorylation of the strand 2-strand 1 hybrid and the subsequent cleavage of double-stranded DNA (dsDNA) by lambda exonuclease, resulting in the release of AuNP-strand 2 conjugates and [Ru(NH 3 ) 6 ] 3+ from the gold electrode surface and consequently the decrease of electrochemical signal. The PNK activity can be simply monitored by the measurement of [Ru(NH 3 ) 6 ] 3+ peak current signal. This assay is very sensitive with a detection limit of as low as 7.762 × 10 -4 UmL -1 and exhibits a large dynamic range from 0.001 to 10UmL -1 . Moreover, this method can be used to screen the PNK inhibitors, and it shows excellent performance in real sample analysis, thus holding great potential for further applications in biological researches and clinic diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Mercaptursäure und Nukleosidaddukt im Harn als Biomarker in 1-Hydroxymethylpyren-exponierten Ratten

NASA Astrophysics Data System (ADS)

Ma, Lan

2002-01-01

1-Methylpyren (MP) ist hepatokanzerogen in neugeborenen männlichen Mäusen. Durch Hydroxylierung an der benzylischen Stelle und anschließende Sulfonierung wird MP zu DNA-reaktivem 1-Sulfooxymethylpyren (SMP) aktiviert. In der Ratte führt die Exposition des benzylischen Alkohols, 1-Hydroxymethylpyren (HMP), zur DNA-Adduktbildung in verschiedenen Geweben. Eventuelle Konsequenz der Toxifizierung ist die Ausscheidung entsprechender Mercaptursäure und Nukleosidaddukt im Harn, welche aufgrund ihrer Herkunft als Biomarker eignen könnten. In dieser Arbeit wird die Ausscheidung der Mercaptursäure und des N2-Desoxyguanosinadduktes in HMP-exponierten Ratten untersucht. Nach der Applikation von HMP bzw. MP wurden weniger als 1 % der Dosis als MPMA über Urin und Faeces ausgeschieden (0 - 48 h). Die Ausscheidung erfolgt hauptsächlich in den ersten 24 h nach der Applikation. MPdG konnte weder in Urin noch in Faeces der HMP-behandelten Tieren identifiziert werden. Nach direkter SMP-Applikation wurde MPdG nur in sehr geringe Menge (weniger als 0,9 ppm in 12 h) im Urin gefunden. Aufgrund der geringen Menge eignet sich MPdG nicht als Biomarker. MPMA dagegen, lässt sich analytisch gut erfassen. Es sollte daher untersucht werden, ob MPMA die Toxifizierung des HMP wiederspiegelt. Die Voraussetzung dafür ist die Kenntnisse über das Metabolismusmuster von HMP. Es wurde daher umfassende Untersuchungen zum Metabolismus des HMP durchgeführt. Die Ergebnisse zeigten, dass mehr als 80 % der Metaboiten in ihrer oxidierten Form (PCS, deren Glucuronsäure-Konjugate sowie phenolische Sulfatester der PCS) ausgeschieden wurden. Demnach spielt die Oxidation des HMP zu PCS eine sehr wichtige Rolle bei der Detoxifizierung und Ausscheidung von HMP. Ferne konnte nachgewiesen werden, dass die Enzyme Alkohol- und Aldehyd-Dehydrogenase an der Oxidation von HMP beteiligt waren. Die Inhibitoren Disulfiram und Ethanol der o. g. Enzyme wurde daher zur Modulation der Detoxifizierung in vivo eingesetzt. Die Veränderungen in der Toxifizierung von HMP zu SMP wurden durch die SMP-Konzentration im Plasma, die DNA-Addukthäufigkeit und die MPMA-Ausscheidung erfasst. Die Vorbehandlung von Disulfiram und Ethanol führte zu tendentielle Erhöhung der SMP-Konzentration im Plasma, DNA-Addukthäufigkeit in der Leber und die MPMA-Ausscheidung. Bemerkenswert ist jedoch, dass bereits eine Dosis von 0,2 g Ethanol/kg Körpermasse bereits zu statistisch signifikanten Erhöhungen der MPMA-Ausscheidung bei weiblichen Ratten. 1-Methylpyrene is hepatocarcinogenic in rodents. It is metabolized primarily to 1-hydroxymethylpyrene (HMP) by various cDNA-expressed rat and human cytochromes P450. HMP is activated to a highly reactive sulfuric acid ester, 1-sulfooxymethylpyrene (SMP), by sulphotransferases. In the rat, this activation pathway leads to the formation of DNA adducts in various tissues. Possible consequences of the toxification could be the excretion of the corresponding mercapturic acid and nucleosidadduct in urine and feces. Because of their origin, these substances should reflex the toxification process may be used as biomarkers. We investigate the excretion of 1-methylpyrenyl-mercapturic acid (MPMA) and the excretion of N2-(1-methylpyrenyl)-desoxyguanosin (MPdG) in urine and feces of HMP-treated rats. These studies showed that only a minor portion (< 1 %) of the administered dose of 1-HMP was excreted as mercapturic acid. MPdG could not be identified in urine and feces of HMP-treated rats. Treating rats with the active spieces sulfooxymethylpyrene, 0.9 ppm of the dose was found excreted within 12 h. I now investigated the alternative metabolic pathways of HMP. More than 50 % of the dose (administered intraperitoneally) was excreted as free 1-pyrenyl carboxylic acid and its glucuronic acid conjugate primarily in the urine. Other major urinary metabolites were phenolic sulpho conjugates of ring-oxidized 1-pyrenyl carboxylic acid (> 30 %). Minor metabolites were phenolic sulpho conjugates of HMP (< 5 %). The glucuronic acid conjugate of HMP was found in very small amounts. In total, > 80 % of the metabolites excreted were oxidized at the exocyclic carbon. This side-chain oxidation, probably catalyzed by alcohol and aldehyde dehydrogenases, appears to represent a detoxification pathway. Indeed, administration of ethanol shortly before the administration of HMP to rats increased the levels of SMP detected in blood, of DNA adducts formed in tissues and of mercapturic acid excreted. These effects were observed even at very low dose levels of ethanol (0.2 g per kg body weight). Similar effects were shown after administration of Disulfiram, an inhibitor of aldehyde dehydrogenase.

Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

PubMed

Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

2018-05-09

The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

Systemic aspects of conjugal resilience in couples with a child facing cancer and marrow transplantation.

PubMed

Martin, Julie; Péloquin, Katherine; Vachon, Marie-France; Duval, Michel; Sultan, Serge

The negative impact of paediatric cancer on parents is well known and is even greater when intensive treatments are used. This study aimed to describe how couples whose child has received a transplant for the treatment of leukaemia view conjugal resilience and to evaluate the role of we-ness as a precursor of conjugal adjustment. Four parental couples were interviewed. Interviews were analysed in two ways: inductive thematic analysis and rating of verbal content with the We-ness Coding Scale . Participants report that conjugal resilience involves the identification of the couple as a team and cohesion in the couple. Being a team generates certain collaborative interactions that lead to conjugal resilience. A sense of we-ness in parents is associated with fluctuation in the frequency of themes. Participants' vision of conjugal resilience introduced novel themes. The sense of we-ness facilitates cohesion and the process of conjugal resilience.

Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

PubMed

Wang, Tieyan; Chen, Qixian; Lu, Hongguang; Li, Wei; Li, Zaifen; Ma, Jianbiao; Gao, Hui

2016-08-17

The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression.

Signal Amplification by Glyco-qPCR for Ultrasensitive Detection of Carbohydrates: Applications in Glycobiology**

PubMed Central

Kwon, Seok Joon; Lee, Kyung Bok; Solakyildirim, Kemal; Masuko, Sayaka; Ly, Mellisa; Zhang, Fuming; Li, Lingyun; Dordick, Jonathan S.; Linhardt, Robert J.

2012-01-01

Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery. PMID:23073897

Nucleotide Sequences and Comparison of Two Large Conjugative Plasmids from Different Campylobacter species

DTIC Science & Technology

2004-01-01

alleles have different predicted lengths, e.g. in pCC31, cpp46 starts with ATGATG whereas in pTet this gene starts with only one ATG; in ssb1 , cmgB7 and...homologues in plasmid pVT745 from Actinobacillus actinomycetemcomitans, and a single-stranded DNA-binding protein ssb1 that may coat the single-stranded

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Phototoxic Activity and DNA Interactions of Water-Soluble Porphyrins and Their Rhenium(I) Conjugates.

PubMed

Mion, Giuliana; Gianferrara, Teresa; Bergamo, Alberta; Gasser, Gilles; Pierroz, Vanessa; Rubbiani, Riccardo; Vilar, Ramon; Leczkowska, Anna; Alessio, Enzo

2015-11-01

In the search for alternative photosensitizers for use in photodynamic therapy (PDT), herein we describe two new water-soluble porphyrins, a neutral fourfold-symmetric compound and a +3-charged tris-methylpyridinium derivative, in which either four or one [1,4,7]-triazacyclononane (TACN) units are connected to the porphyrin macrocycle through a hydrophilic linker; we also report their corresponding tetracationic Re(I) conjugates. The in vitro (photo)toxic effects of the compounds toward the human cell lines HeLa (cervical cancer), H460M2 (non-small-cell lung carcinoma), and HBL-100 (non-tumorigenic epithelial cells) are reported. Three of the compounds are not cytotoxic in the dark up to 100 μm, and the fourfold-symmetric couple revealed very good phototoxic indexes (PIs). The intracellular localization of all derivatives was studied in HeLa cells by confocal fluorescence microscopy. Although low nuclear localization was observed for some of them, it still prompted us to investigate their capacity to bind both quadruplex and duplex DNA; we observed significant selectivity in the tris-methylpyridinium derivatives for G-quadruplex interactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and electrochemical response of DNA functionalized 2nm gold nanoparticles confined in a nanochannel array.

PubMed

Peinetti, Ana S; Ceretti, Helena; Mizrahi, Martín; González, Graciela A; Ramírez, Silvana A; Requejo, Félix G; Montserrat, Javier M; Battaglini, Fernando

2018-06-01

Polyvalent gold nanoparticle oligonucleotide conjugates are subject of intense research. Even though 2nm diameter AuNPs have been previously modified with DNA, little is known about their structure and electrochemical behavior. In this work, we examine the influence of different surface modification strategies on the interplay between the meso-organization and the molecular recognition properties of a 27-mer DNA strand. This DNA strand is functionalized with different sulfur-containing moieties and immobilized on 2nm gold nanoparticles confined on a nanoporous alumina, working the whole system as an electrode array. Surface coverages were determined by EXAFS and the performance as recognition elements for impedance-based sensors is evaluated. Our results prove that low DNA coverages on the confined nanoparticles prompt to a more sensitive response, showing the relevance in avoiding the DNA strand overcrowding. The system was able to determine a concentration as low as 100pM of the complementary strand, thus introducing the foundations for the construction of label-free genosensors at the nanometer scale. Copyright © 2018 Elsevier B.V. All rights reserved.

CRISPR-Cas9 Structures and Mechanisms.

PubMed

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

PubMed

Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

2015-10-01

The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector.

PubMed

Guo, Xin Dong; Tandiono, Fanny; Wiradharma, Nikken; Khor, Dingyue; Tan, Chuan Guan; Khan, Majad; Qian, Yu; Yang, Yi-Yan

2008-12-01

Cholesterol-conjugated H(5)R(10) and H(10)R(10) oligopeptides (HR15-Chol and HR20-Chol) were designed and synthesized. These amphiphilic oligopeptides were able to self-assemble into cationic micelles in aqueous solution at low concentrations, and their critical micelle concentrations in sodium acetate buffer (20mM, pH 5.0) were 17.8 and 28.2mg/L respectively. The micelle formation was further evidenced via SEM and dynamic light scattering analyses. The average hydrodynamic size of HR15-Chol and HR20-Chol micelles was about 425 and 435 nM in diameter with zeta potential of 64 and 66 mV respectively. The formation of micelles increased local concentration of cationic charge, leading to higher DNA binding efficiency as compared to the control peptides HR15 and HR20. The minimum size observed for HR15-Chol/DNA and HR20-Chol/DNA complexes was about 175-176 nM, and the maximum zeta potential was around 61-62 mV. In comparison, HR15 and HR20 formed DNA complexes with a similar size but significantly lower zeta potential (i.e. about 31-40 mV). In particular, after being challenged by DMEM medium, the size of peptide/DNA complexes was increased significantly and their surface charge was neutralized. Nevertheless, the size of the micelle/DNA complexes formed from HR15-Chol and HR20-Chol was still about 200 nM with positive charge of around 20 mV at high N/P ratios. The micelles induced much higher overall gene expression (i.e. luciferase expression) levels than the peptides in both HepG2 and HEK293 cell lines. Increasing the histidine residue from 0 to 5 to 10 further increased gene expression efficiency. In particular, HR20-Chol micelles yielded 95% GFP-positive HepG2 cells at N/P 50, much higher than that induced by PEI at its optimal N/P ratio (i.e. 10), which was 6.8%. In 4T1 cells, HR20-Chol induced 2 times higher luciferase expression level than PEI at their optimal N/P ratios. Moreover, HR20-Chol micelle/DNA complexes were less cytotoxic than PEI/DNA complexes. These micelles may be a promising carrier for delivery of therapeutic genes.